Mammalian telomerase

ABSTRACT

Nucleic acids comprising the RNA component of a mammalian telomerase are useful as pharmaceutical, therapeutic, and diagnostic reagents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.08/472,802, filed Jun. 7, 1995, now U.S. Pat. No. 5,958,680, which is acontinuation-in-part of U.S. patent application Ser. No. 08/330,123,filed Oct. 27, 1994 (U.S. Pat. No. 5,583,016), which is acontinuation-in-part of U.S. patent application Ser. No. 08/272,102,filed Jun. 7, 1994 (abandoned), each of which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to human telomerase, a ribonucleoproteinenzyme involved in human telomere DNA synthesis. The invention providesmethods and compositions relating to the fields of molecular biology,chemistry, pharmacology, and medical and diagnostic technology.

2. Description of Related Disclosures

The DNA at the ends or telomeres of the chromosomes of eukaryotesusually consists of tandemly repeated simple sequences. Telomerase is aribonucleoprotein enzyme that synthesizes one strand of the telomericDNA using as a template a sequence contained within the RNA component ofthe enzyme. See Blackburn, 1992, Annu. Rev. Biochem. 61:113-129,incorporated herein by reference.

The RNA component of human telomerase has not been reported in thescientific literature to date, although human telomerase is known tosynthesize telomeric repeat units with the sequence 5′-TTAGGG-3′. SeeMorin, 1989, Cell 59:521-529, and Morin, 1991, Nature 353:454-456,incorporated herein by reference. This knowledge has not been sufficientto enable the isolation and identification of the remainder of thenucleotide sequence of the RNA component of human telomerase. The RNAcomponent of the telomerase enzymes of Saccharomyces cerevisiae, certainspecies of Tetrahymena, as well as that of other ciliates, such asEuplotes and Glaucoma, has been sequenced and reported in the scientificliterature. See Singer and Gottschling, Oct. 21, 1994, Science266:404-409; Lingner et al., 1994, Genes & Development 8:1984-1988;Greider and Blackburn, 1989, Nature 337:331-337; Romero and Blackburn,1991, Cell 67:343-353; and Shippen-Lentz and Blackburn, 1990, Science247:546-552, each of which is incorporated herein by reference. Thetelomerase enzymes of these ciliates synthesize telomeric repeat unitsdistinct from that in humans.

There is a great need for more information about human telomerase.Despite the seemingly simple nature of the repeat units of telomericDNA, scientists have long known that telomeres have an importantbiological role in maintaining chromosome structure and function. Morerecently, scientists have speculated that loss of telomeric DNA may actas a trigger of cellular senescence and aging and that regulation oftelomerase may have important biological implications. See Harley, 1991,Mutation Research 256:271-282, incorporated herein by reference.

Methods for detecting telomerase activity, as well as for identifyingcompounds that regulate or affect telomerase activity, together withmethods for therapy and diagnosis of cellular senescence andimmortalization by controlling telomere length and telomerase activity,have also been described. See PCT patent publication No. 93/23572,published Nov. 25, 1993, and U.S. patent application Ser. Nos.08/315,216 inventors Michael D. West, Jerry Shay, and Woodring Wright,filed Sep. 28, 1994; 08/315,214 inventors Nam Woo Kim, Scott Weinrich,and Calvin B. Harley, filed Sep. 28, 1994; 08/288,501, filed Aug. 10,1994; 08/014,838, filed Feb. 8, 1993; 08/153,051 and 08/151,477, eachfiled Nov. 12, 1993; 08/060,952, filed May 13, 1993; 08/038,766, filedMar. 24, 1993; and 07/882,438, filed May 13, 1992, each of which isincorporated herein by reference.

Significant improvements to and new opportunities fortelomerase-mediated therapies and telomerase assays and screeningmethods could be realized if nucleic acid comprising the RNA componentand/or encoding the protein components of telomerase were available inpure or isolatable form and the nucleotide sequences of such nucleicacids were known. The present invention meets these and other needs andprovides such improvements and opportunities.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides the RNA component of,as well as the gene for the RNA component of, human telomerase insubstantially pure form, as well as nucleic acids comprising all or atleast a useful portion of the nucleotide sequence of the RNA componentof human telomerase. The present invention also provides RNA componentnucleic acids from other species, which nucleic acids share substantialhomology with the RNA component of human telomerase, including but notlimited to, the RNA components of mammals, such as primates. Otheruseful nucleic acids of the invention include nucleic acids withsequences complementary to the RNA component; nucleic acids withsequences related to but distinct from nucleotide sequences of the RNAcomponent and which interact with the RNA component or the gene for theRNA component or the protein components of human telomerase in a usefulway; and nucleic acids that do not share significant sequence homologyor complementarity to the RNA component or the gene for the RNAcomponent but act on the RNA component in a desired and useful way. Asdescribed more fully below, the nucleic acids of the invention includeboth DNA and RNA molecules and modified analogues of either and serve avariety of useful purposes.

Thus, one type of useful nucleic acid of the invention is an antisenseoligonucleotide, a triple helix-forming oligonucleotide, or otheroligonucleotide or oligonucleotide mimetic (e.g., antisense PNA) thatcan be used in vivo or in vitro to inhibit the activity of humantelomerase. Such oligonucleotides can block telomerase activity in anumber of ways, including by preventing transcription of the telomerasegene (for instance, by triple helix formation) or by binding to the RNAcomponent of telomerase in a manner that prevents a functionalribonucleoprotein telomerase from assembling or prevents the RNAcomponent, once assembled into the telomerase enzyme complex, fromserving as a template for telomeric DNA synthesis. Typically, anddepending on mode of action, these oligonucleotides of the inventioncomprise a specific sequence of from about 10 to about 25 to 200 or morenucleotides that is either identical or complementary to a specificsequence of nucleotides in the RNA component of telomerase or the genefor the RNA component of telomerase.

Another type of useful nucleic acid of the invention is a ribozyme ableto cleave specifically the RNA component of human telomerase, renderingthe enzyme inactive. Yet another type of useful nucleic acid of theinvention is a probe or primer that binds specifically to the RNAcomponent of human telomerase and so can be used, e.g., to detect thepresence of telomerase in a sample. Finally, useful nucleic acids of theinvention include recombinant expression plasmids for producing thenucleic acids of the invention. One especially useful type of such aplasmid is a plasmid used for human gene therapy. Useful plasmids of theinvention for human gene therapy come in a variety of types, includingnot only those that encode antisense oligonucleotides or ribozymes butalso those that drive expression of the RNA component of humantelomerase or a deleted or otherwise altered (mutated) version of theRNA component of human (or other species with RNA component sequencessubstantially homologous to the human RNA component) telomerase or thegene for the same.

In a second aspect, the invention provides methods for treating acondition associated with the telomerase activity within a cell or groupof cells by contacting the cell(s) with a therapeutically effectiveamount of an agent that alters telomerase activity in that cell. Suchagents include the telomerase RNA component-encoding nucleic acids,triple helix-froming oligonucleotides, antisense oligonucleotides,ribozymes, and plasmids for human gene therapy described above. In arelated aspect, the invention provides pharmaceutical compositionscomprising these therapeutic agents together with a pharmaceuticallyacceptable carrier or salt.

In a third aspect, the invention provides diagnostic methods fordetermining the level, amount, or presence of the RNA component of humantelomerase, telomerase, or telomerase activity in a cell, cellpopulation, or tissue sample, or an extract of any of the foregoing. Ina related aspect, the present invention provides useful reagents forsuch methods (including the primers and probes noted above), optionallypackaged into kit form together with instructions for using the kit topractice the diagnostic method.

In a fourth aspect, the present invention provides recombinanttelomerase preparations and methods for producing such preparations.Thus, the present invention provides a recombinant human telomerase thatcomprises the protein components of human telomerase as well as theprotein components of telomerase from a mammalian species with an RNAcomponent substantially homologous to the RNA component of humantelomerase in association with a recombinant RNA component of theinvention. Such recombinant RNA component molecules of the inventioninclude those that differ from naturally occurring RNA componentmolecules by one or more base substitutions, deletions, or insertions,as well as RNA component molecules identical to a naturally occurringRNA component molecule that are produced in recombinant host cells. Themethod for producing such recombinant telomerase molecules comprisestransforming a eukaryotic host cell that expresses the proteincomponents of telomerase with a recombinant expression vector thatencodes an RNA component molecule of the invention, and culturing saidhost cells transformed with said vector under conditions such that theprotein components and RNA component are expressed and assemble to forman active telomerase molecule capable of adding sequences (notnecessarily the same sequence added by native telomerase) to telomeresof chromosomal DNA.

In a fifth aspect, the invention provides methods for purifying theprotein components of human telomerase as well as the protein componentsof telomerase from a mammalian species with an RNA componentsubstantially homologous to the RNA component of human telomerase. Thepresent invention also provides methods for isolating and identifyingnucleic acids encoding such protein components. In related aspects, thepresent invention provides purified human telomerase and purifiedtelomerase of mammalian species with an RNA component substantiallyhomologous to the RNA component of human telomerase, as well as purifiednucleic acids that encode one or more components of such telomerasepreparations. The present invention also provides pharmaceuticalcompositions comprising as an active ingredient the protein componentsof telomerase or a nucleic acid that encodes or interacts with a nucleicacid that encodes a protein component of telomerase.

The present invention also provides a method for diagnosing a disease(e.g., neoplasia) in a human patient, wherein a diagnostic assay (e.g.,determination of hTR) is used to determine if a predeterminedpathognomonic concentration of hTR RNA is present in cells in abiological sample from a human patient; if the assay indicates thepresence of a pathogonomonic amount of hTR outside of the normal range(e.g., beyond the predetermined pathognomonic concentration), thepatient is diagnosed as having a disease condition or predisposition.

In a variation of the invention, polynucleotides of the invention areemployed for diagnosis of pathological conditions or genetic diseasethat involve neoplasia, aging, or other medical conditions related totelomerase function, and more specifically conditions and diseases thatinvolve alterations in the structure or abundance of a hTR RNA of hTRgene sequence, or which are linked to a pathognomonic hTR allele whichcan be detected by RFLP and/or allele-specific PCR, or other suitabledetection method.

The invention also provides antisense polynucleotides complementary tohTR polynucleotide sequences, typically complementary to polynucleotidesequences which are substantially identical to a naturally-occurringmammalian hTR gene sequence. Such antisense polynucleotides are employedto inhibit transcription and/or stability and/or functionality of thehTR RNA species and thereby effect a reduction in the amount of therespective telomerase activity in a cell (e.g., a neoplastic cell of apatient). Such antisense polynucleotides can function astelomerase-modulating agents by inhibiting the formation of functional(catalytically active and high fidelity) telomerase holoenzyme requiredfor correct telomere replication and repair in a cell. Antisensepolynucelotides can be combined with other antineoplastic therapeuticmodalities, such as ionizing radiation or chemotherapy (e.g., with aDNA-damaging agent such as bleomycin, cisplatin, nitrogen mustard,doxyrubicin, nucleotide analogs, and the like). The antisensepolynucleotides can promote cell death in susceptible cells (e.g.,replicating cells requiring telomerase activity for DNA repair orreplication). The hTR antisense polynucleotides are substantiallyidentical to at least 25 contiguous nucleotides of the complementarysequence of the hTR RNA sequence disclosed herein. The hTR antisensepolynucleotides are typically ssDNA, ssRNA, methylphosphonate backbonenucleic acids, phosphorothiolate backbone, polyamide nucleic acids, andthe like antisense structures known in the art. In one aspect of theinvention, an antisense polynucleotide is administered to inhibittranscription and/or activity of of hTR and telomerase in a cell, suchas a replicable human cell.

The invention also provides hTR polynucleotide probes for diagnosis ofdisease states (e.g., neoplasia or preneoplasia) by detection of a hTRRNA or hTR gene rearrangements or amplification of the hTR gene in cellsexplanted from a patient, or detection of a pathognomonic hTR allele(e.g., by RFLP or allele-specific PCR analysis). Typically, thedetection will be by in situ hybridization using a labeled (e.g., ³²P,³⁵S, ¹⁴C, ³H, fluorescent, biotinylated, digoxigeninylated) antisensepolynucleotide complementary to hTR, although Northern blotting, dotblotting, or solution hybridization on bulk RNA or poly A⁺ RNA isolatedfrom a cell sample may be used, as may PCR amplification usinghTR-specific primers. Cells which contain an altered amount (typically asignificant increase) of hTR RNA as compared to non-neoplastic cells ofthe same cell type(s) will be identified as candidate diseased cells.Similarly, the detection of pathognomonic rearrangements oramplification of the hTR gene locus or closely linked loci in a cellsample will identify the presence of a pathological condition or apredisposition to developing a pathological condition (e.g., cancer,genetic disease). The polynucleotide probes are also used for forensicidentification of individuals, such as for paternity testing oridentification of criminal suspects or unknown decedents.

The present invention also provides a method for diagnosing a disease(e.g., neoplasia) in a human patient, wherein a diagnostic assay (e.g.,in situ polynucleotide hybridization of fixed cells by a labelled hTRprobe that specifically binds human hTR RNA or gene sequences) is usedto determine if a predetermined pathognomonic concentration of hTR RNAis present in a biological sample from a human patient; if the assayindicates the presence of hTR RNA outside of the normal range (e.g.,beyond the predetermined pathognomonic concentration), the patient isdiagnosed as having a disease condition or predisposition.

The invention also provides therapeutic agents which inhibit neoplasiaor apoptosis by modulating telomerase function by inhibiting oraugmenting formation of hTR RNA; such agents can be used aspharmaceuticals. Such pharmaceuticals will be used to treat a variety ofhuman and veterinary diseases, such as: neoplasia, hyperplasia,neurodegenerative diseases, aging, AIDS, fungal infection, and the like.In an embodiment, the agent consists of a gene therapy vector capable oftranscribing a hTR RNA sequence or its complement, or alternatively anenzymatically inactive hTR RNA which can competitively inhibit formationof functional telomerase holoenzyme.

Other features and advantages of the invention will be apparent from thefollowing description of the drawings, preferred embodiments of theinvention, the examples, and the claims.

Definitions

The term “hTR polynucleotide” as used herein refers to a polynucleotideof at least 20 nucleotides wherein the polynucleotide comprises asegment of at least 20 nucleotides which: are at least 85 percentidentical to a naturally-occurring hTR RNA sequence Some hTRpolynucleotides having sequence variations as compared to anaturally-occurring hTR sequence can be suitable as hybridizationprobes, PCR primers, LCR amplimers, and the like.

The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence. In contradistinction, the term “complementary to” is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence “TATAC” corresponds to a reference sequence “TATAC”and is complementary to a reference sequence “GTATA”.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “comparisonwindow”, “sequence identity”, “percentage of sequence identity”, and“substantial identity”. A “reference sequence” is a defined sequenceused as a basis for a sequence comparison; a reference sequence may be asubset of a larger sequence, for example, as a segment of a full-lengthhTR gene sequence. Generally, a reference sequence is at least 20nucleotides in length, frequently at least 25 nucleotides in length, andoften at least 50 nucleotides in length. Since two polynucleotides mayeach (1) comprise a sequence (i.e., a portion of the completepolynucleotide sequence) that is similar between the twopolynucleotides, and (2) may further comprise a sequence that isdivergent between the two polynucleotides, sequence comparisons betweentwo (or more) polynucleotides are typically performed by comparingsequences of the two polynucleotides over a “comparison window” toidentify and compare local regions of sequence similarity.

A “comparison window”, as used herein, refers to a conceptual segment ofat least 25 contiguous nucleotide positions wherein a polynucleotidesequence may be compared to a reference sequence of at least 25contiguous nucleotides and wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by the local homology algorithm ofSmith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988)Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection, and the bestalignment (i.e., resulting in the highest percentage of homology overthe comparison window) generated by the various methods is selected.

The term “sequence identity” means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. The terms “substantial identity” as used hereindenotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence that has at least 80 percentsequence identity, preferably at least 85 percent identity and often 89to 95 percent sequence identity, more usually at least 99 percentsequence identity as compared to a reference sequence over a comparisonwindow of at least 20 nucleotide positions, frequently over a window ofat least 30-50 nucleotides, wherein the percentage of sequence identityis calculated by comparing the reference sequence to the polynucleotidesequence which may include deletions or additions which total 20 percentor less of the reference sequence over the window of comparison. Thereference sequence may be a subset of a larger sequence, for example, asa segment of the full-length hTR gene sequence as disclosed herein.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotinyl moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcalorimetric methods). Various methods of labeling polypeptides andglycoproteins are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following;radioisotopes. (e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I, ¹³¹I), fluorescent labels(e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g.,horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase), biotinyl groups, predetermined polypeptide epitopesrecognized by a secondary reporter (e.g., leucine zipper pair sequences,binding sites for secondary antibodies, transcriptional activatorpolypeptide, metal binding domains, epitope tags). In some embodiments,labels are attached by spacer arms of various lengths to reducepotential steric hindrance.

As used herein the terms “pathognomonic concentration”, “pathognomonicamount”, and “pathognomonic hybridization pattern” refer to aconcentration, amount, or localization pattern, respectively, of a hTRmRNA in a sample, that indicates the presence of a pathological (e.g.,neoplastic, senescent, immunodeficient, neurodegenerative, inflammatory,etc.) condition or a predisposition to developing a neoplastic disease,such as carcinoma, sarcoma, or leukemia. A pathognomonic amount is anamount of hTR RNA in a cell or cellular sample that falls outside therange of normal clinical values that is established by prospectiveand/or retrospective statistical clinical studies. Generally, anindividual having a neoplastic disease (e.g., carcinoma, sarcoma, orleukemia) will exhibit an amount of hTR RNA in a cell or tissue samplethat is outside the range of concentrations that characterize normal,undiseased individuals; typically the pathognomonic concentration is atleast about one standard deviation outside the mean normal value, moreusually it is at least about two standard deviations or more above themean normal value. However, essentially all clinical diagnostic testsproduce some percentage of false positives and false negatives. Thesensitivity and selectivity of the diagnostic assay must be sufficientto satisfy the diagnostic objective and any relevant regulatoryrequirements. In general, the diagnostic methods of the invention areused to identify individuals as disease candidates, providing anadditional parameter in a differential diagnosis of disease made by acompetent health professional.

As used herein, the term “disease allele” refers to an allele of a genewhich is capable of producing a recognizable disease. A disease allelemay be dominant or recessive and may produce disease directly or whenpresent in combination with a specific genetic background orpre-existing pathological condition. A disease allele may be present inthe gene pool or may be generated de novo in an individual by somaticmutation.

The term “antineoplastic agent” is used herein to refer to agents thathave the functional property of inhibiting a development or progressionof a neoplasm in a human, often including inhibition of metastasis ormetastatic potential.

As used herein, the term “operably linked” refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is“operably linked” when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. Operably linked means that the DNA sequences beinglinked are typically contiguous and, where necessary to join two proteincoding regions, contiguous and in reading frame. However, sinceenhancers generally function when separated from the promoter by severalkilobases and intronic sequences may be of variable lengths, somepolynucleotide elements may be operably linked but not contiguous. Astructural gene (e.g., a HSV tk gene) which is operably linked to apolynucleotide sequence corresponding to a transcriptional regulatorysequence of an endogenous gene is generally expressed in substantiallythe same temporal and cell type-specific pattern as is thenaturally-occurring gene.

As used herein, the term “transcriptional unit” or “transcriptionalcomplex” refers to a polynucleotide sequence that comprises a structuralgene (exons), a cis-acting linked promoter and other cis-actingsequences necessary for efficient transcription of the structuralsequences, distal regulatory elements necessary for appropriatetissue-specific and developmental transcription of the structuralsequences, and additional cis sequences important for efficienttranscription and translation (e.g., polyadenylation site, mRNAstability controlling sequences).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Telomerase activity from cells expressing template-mutated TRC3DEAE-sepharose fractionated extracts from mutant TRC-3-expressing stabletransformants were assayed for telomerase activity using conventionalassays under various reaction conditions. Extracts from cells expressingMuC+17 TRC-3 (lanes 1, 4, 7, 10, 13, 16 labeled C*), MuC TRC-3 (lanes 2,5, 8, 11, 14, 17 labeled C), or MuA TRC-3 (lanes 3, 6, 9, 12, 15, 18labeled A) were assayed under normal reaction conditions (lanes 1-6),normal plus 0.5 mM ddCTP (lanes 7-9), normal minus dTTP plus 0.5 mMddTTP (lanes 10-12), or normal minus DATP plus 0.5 mM ddATP (lanes13-18). Assay reactions in lanes 1-9 contained 8 μM total dGTP, 1 μM ofwhich was ³²P-dGTP (800 Ci/mmol). To facilitate mutant telomerasedetection, assay reactions in lanes 10-18 contained 8 μM total dGTP, 2μM of which was ³²P-dGTP (800 Ci/mmol). Extracts were treated withDNase-free RNase (25 μg/ml for 10 min at 30° C.) prior to telomeraseassays (lanes 1-3, 16-18). Flanking lanes contain DNA markers with sizesin nucleotides (nt) as indicated.

FIG. 2A. The steady-state level of hTR and GAPDH RNA was determinedusing quantitative RT-PCR. Controls show that all PCR quantitations werein the linear range up to 25 cycles. RT-PCR was analyzed for five normaltelomerase-negative cell lines (1-5) and five tumor telomerase-positivecells lines (6-10): 1) Primary fetal lung; 2) Primary fetal hand skin;3) Adult primary prostate; 4) Primary sinovial fibroblasts; 5) Foreskinfibroblasts; 6) Melanoma LOX; 7) Leukemia U25x; 8) NCIH23 lungcarcinoma; 9) Colon tumor SW620; 10) Breast tumor MCF7. PCR productswere labeled with ³²P, resolved on a 6% PAGE, and quantified using aPhosphorImager. The relative transcription is expressed in arbitraryunits.

FIG. 2B. Northern blot of hTR RNA prepared from human tissues. Total 293RNA was prepared by guanidinium thiocyanate-phenol/chloroform extractionand tissue RNAs were obtained from Clonetech. Thirty micrograms of totalRNA was loaded onto a 1.5% agarose/formaldehyde gel and transferred ontoHybond N. The blot was probed with the human telomerase RNA in Churchhybridization solution. Telomerase RNA was detected in all tissues, withthe highest level of expression seen in the testes and ovary. As acontrol for loading, the blot was washed and re-probed for 18S rRNA.

FIG. 2C. Histogram of the relative levels of telomerase RNA in differenttissues. The Northern hybridization signals were quantified on aPhosphorImager and the hTR was normalized to the 18S loading control.The relative signal is expressed as arbitrary units.

FIG. 3. Mean TRF length in hTR antisense and vector control cells. HeTe7cells that stably express 10-3-hTR antisense or vector control wereselected in hygromycin and puromycin media and harvested at 23 PDL posttransfection. Nuclear DNA was purified, cut with HinfI and RsaI, and runon a 0.5% agarose gel The DNA was probed in the gel with a (TTAGGG)₃(SEQ ID NO:26) oligonuceotide to label the telomeric terminalrestriction fragments (TRF). The gel was scanned with a MolecularDynamic's PhosphorImager and the mean TRF quantitated as described(Allsopp et al. (1992) Proc. Natl. Acad. Sci. (USA) 89:10114). Thedashed lines indicate the average mean TRF for the antisense and controlgroups.

FIG. 4 Schematic representation of in situ PCR method.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides methods, reagents, and pharmaceuticalcompositions relating to the ribonucleoprotein human telomerase. Theinvention in part arises out of the cloning and isolation of the RNAcomponent of human telomerase and the gene for that RNA component. Thenucleotide sequence of the RNA component of human telomerase is shownbelow. For convenience, the sequence is shown using the standardabbreviations for ribonucleotides (A is riboadenine, G is riboguanine, Cis ribocytidine, and U is uridine). Those of skill in the art recognizethat the sequence shown below (SEQ ID NO:1) also shows the sequence ofthe cDNA, in which the ribonucleotides are replaced bydeoxyribonucleotides (with uridine being replaced by thymidine).

                                                     50 GGGUUGCGGAGGGUGGGCCU GGGAGGGGUG GUGGCCAUUU UUUGUCUAAC                                                     100 CCUAACUGAGAAGGGCGUAG GCGCCGUGCU UUUGCUCCCC GCGCGCUGUU                                                     150 UUUCUCGCUGACUUUCAGCG GGCGGAAAAG CCUCGGCCUG CCGCCUUCCA                                                     200 CCGUUCAUUCUAGAGCAAAC AAAAAAUGUC AGCUGCUGGC CCGUUCGCCC                                                     250 CUCCCGGGGACCUGCGGCGG GUCGCCUGCC CAGCCCCCGA ACCCCGCCUG                                                     300 GAGGCCGCGGUCGGCCCGGG GCUUCUCCGG AGGCACCCAC UGCCACCGCG                                                     350 AAGAGUUGGGCUCUGUCAGC CGCGGGUCUC UCGGGGGCGA GGGCGAGGUU                                                     400 CAGGCCUUUCAGGCCGCAGG AAGAGGAACG GAGCGAGUCC CCGCGCGCGG                                                     450 CGCGAUUCCCUGAGCUGUGG GACGUGCACC CAGGACUCGG CUCACACAUG                                                     500 CAGUUCGCUUUCCUGUUGGU GGGGGGAACG CCGAUCGUGC GCAUCCGUCA                                                     550 CCCCUCGCCGGCAGUGGGGG CUUGUGAACC CCCAAACCUG ACUGACUGGG          560 CCAGUGUGCU

The sequence above is shown in the 5′-3′ direction and is numbered forreference. The template sequence of the RNA component is believed to belocated within the region defined by nucleotides 50-60(5′-CUAACCCUAAC-3′) (SEQ ID NO:2), which is complementary to a telomericsequence composed of about one-and-two-thirds telomeric repeat units.

This sequence was derived from cDNA clones and from the genomic clone ofthe RNA component. When the RNA component is first transcribed from thecorresponding gene, at least some of the RNA transcripts produced aremuch longer than the ^(˜)560 nucleotide sequence shown above and in factmay comprise more than 1000 nucleotides. However, a fully functionaltelomerase molecule can be assembled from transcripts consisting of the^(˜)560 nucleotide sequence shown above. The 3′-end of the RNA componentin native telomerase is believed to lie within the region defined bynucleotides 514-559 in the sequence above; one analysis suggests thatthe 3′-end may be the U residue at nucleotide 538. Recombinant RNAcomponent molecules comprising less than nucleotides 1-559 of thesequence shown above can also be used to prepare active telomerase.

The cloning of the RNA component of human telomerase required a novelmethod involving negative selection and cycles of positive selection,described below. Initially, however, an attempt was made to clone theRNA component using reverse transcription and a method for cloning theends of cDNA called “5′-RACE” PCR amplification. The reversetranscription reaction was initiated with a primer identical to therepeat unit in the single-strand portion of human telomeric DNA and thuscomplementary to a sequence believed to be present in the RNA componentof human telomerase. The primer also comprised, at its 5′-end, asequence corresponding to a restriction enzyme recognition site.However, when the cDNA produced by the reverse transcription reactionand PCR amplification was examined by gel electrophoresis and nucleotidesequence analysis of the bands of nucleic acid present in the gel, onlyribosomal RNA sequences were detected. Similar problems were encounteredwhen variations of this 5′-RACE approach were attempted using nestedprimers.

The successful cloning effort began with the preparation of cDNA frompurified preparations of human telomerase as well as from cell linesthat have human telomerase activity and from cell lines that do not havedetectable human telomerase activity. The method used to prepare thecDNA is described in detail in Example 1, below. Two negative selectionsteps and successive cycles of positive selection were used inconjunction with the cDNA preparations from the two human cell lines tolower the concentration of unwanted sequences and to raise theconcentration of the desired RNA component sequences.

The negative selection steps involved the preparation of biotinylatedPCR product from cDNA prepared from a human cell line that does not havedetectable telomerase activity. The biotinylated PCR product wasdenatured and then rehybridized in a solution comprising a much lowerconcentration of non-biotinylated PCR product (100 biotinylatedproduct:1 non-biotinylated product) from cDNA prepared from a human cellline that does have telomerase activity. Given the possibility that thetelomerase negative cell line might contain some low amount of the RNAcomponent, the hybridization step was conducted to discriminate orselect against only RNA expressed abundantly in both cell lines. Afterhybridization to a C_(o)t selected to allow hybridization of the mostabundantly expressed RNA, the unwanted material was removed by bindingto streptavidinylated magnetic particles; the supernatant remainingafter particle collection contained the desired cDNA for the RNAcomponent of human telomerase. The process for PCR amplification of cDNAis described in Example 2, below.

This material was further enriched for the desired cDNA by successivecycles of positive selection. In the positive selection step, abiotinylated probe complementary to the predicted template sequence inthe RNA component of human telomerase was hybridized to PCR product froman enriched (by negative selection) sample of the PCR-amplified cDNAfrom a human cell line that has telomerase activity. Afterhybridization, the probe/target complexes were bound to avidinylatedmagnetic beads, which were then collected and used as a source ofnucleic acid enriched in RNA component sequences in further cycles ofpositive selection. The positive selection process is described in moredetail in Examples 3 and 4, below.

After the third cycle of positive selection, the amplification productswere separated by gel electrophoresis, and sections of the gelcorresponding to nucleic acids ^(˜)200 bp in size were removed. Thenucleic acids were then eluted from the gel sections and amplified byPCR. The PCR amplification products were digested with restrictionenzyme NotI and then inserted by ligation into the NotI site of plasmidpBluescriptIISK+, commercially available from Stratagene. The resultingplasmids were transformed into E. coli host cells, and individualcolonies were isolated and used as a source of nucleic acid for furtheranalysis and DNA sequencing. Individual colonies were grown in the wellsof a 96-well microtiter plate, which was then used as a master plate,and blots of DNA from the colonies in the plate were prepared andhybridized to a probe comprising a telomeric repeat sequence andtherefore complementary to the RNA component of human telomerase. Anumber of clones positive by this test were then analyzed by DNAsequencing and a variety of other tests.

These other tests included the following: (1) determination whetherantisense oligonucleotides complementary to the putative RNA componentwould inhibit telomerase activity in human cell extracts known tocontain telomerase; (2) determination whether PCR primers specific for aputative RNA component clone sequence could be used to amplify a nucleicacid present in a telomerase sample and whether the product observed, ifany, would track telomerase activity during purification of telomerase;and (3) determination whether PCR primers specific for a putative RNAcomponent clone sequence could be used to amplify a nucleic acid presentin greater abundance in cell extracts from cells in which telomeraseactivity is known to be high (i.e., tumor cells) than in cell extractsfrom cells known to produce no or only low amounts of telomeraseactivity. One clone, designated plasmid pGRN7, produced results in thesetests consistent with the determination that the plasmid comprised cDNAcorresponding to the RNA component of human telomerase.

Thus, antisense oligonucleotides corresponding to sequences of theputative RNA component sequence of pGRN7 exhibited inhibition oftelomerase activity in vitro. Likewise, when telomerase was purifiedfrom cell extracts by a process involving (1) DEAE chromatography; (2)Sephadex S300 chromatography; and (3) either glycerol gradient, SPsepharose, or phenyl sepharose separation and fractions collected, PCRprimers specific for the putative RNA component sequence of pGRN7amplified a nucleic acid of the appropriate size, and the amount ofamplification product correlated well with the amount of telomeraseactivity observed in the fractions collected. Finally, cell extractsfrom normal (no detectable telomerase activity) and cancer (telomeraseactivity present), as well as testis (telomerase activity present),cells showed corresponding amounts of PCR product upon reversetranscription and PCR amplification (RT-PCR) with primers specific forthe putative RNA component comprised in pGRN7. The protocol for theRT-PCR is described in Examples 5 and 6, below.

The above results provided convincing evidence that the RNA component ofhuman telomerase had been cloned, so plasmid pGRN7 was then used toisolate a genomic clone for the RNA component from a human cell line, asdescribed in Example 7, below. The genomic clone was identified in andisolated from a genomic library of human DNA inserted into a lambdavector FIXII purchased from Stratagene. The desired clone comprising theRNA component gene sequences contained an ^(˜)15 kb insert and wasdesignated clone 28-1. This clone has been deposited with the AmericanType Culture Collection and is available under the accession No. ATCC75925. Lambda clone 28-1 was deposited under the Budapest Treaty on Oct.25, 1994 at the American Type Culture Collection, Rockville, Md. 20852.Various restriction fragments were subcloned from this phage andsequenced. The gene has also been localized to the distal end of the qarm of chromosome 3. The sequence information obtained from a SauIIIA1restriction enzyme recognition site at one end of the ^(˜)15 kb insertto an internal HindIII restriction enzyme recognition site, whichcomprises all of the mature RNA component sequence as well astranscription control elements of the RNA component gene, of lambdaclone 28-1 is shown below (SEQ ID NO:3) using the standarddeoxyribonucleotide abbreviations and depicted in the 5′-3′ direction.

                                                     50 GATCAGTTAGAAAGTTACTA GTCCACATAT AAAGTGCCAA GTCTTGTACT                                                     100 CAAGATTATAAGCAATAGGA ATTTAAAAAA AGAAATTATG AAAACTGACA                                                     150 AGATTTAGTGCCTACTTAGA TATGAAGGGG AAAGAAGGGT TTGAGATAAT                                                     200 GTGGGATGCTAAGAGAATGG TGGTAGTGTT GACATATAAC TCAAAGCATT                                                     250 TAGCATCTACTCTATGTAAG GTACTGTGCT AAGTGCAATA GTGCTAAAAA                                                     300 CAGGAGTCAGATTCTGTCCG TAAAAAACTT TACAACCTGG CAGATGCTAT                                                     35O GAAAGAAAAAGGGGATGGGA GAGAGAGAAG GAGGGAGAGA GATGGAGAGG                                                     400 GAGATATTTTACTTTTCTTT CAGATCGAGG ACCGACAGCG ACAACTCCAC                                                     450 GGAGTTTATCTAACTGAATA CGAGTAAAAC TTTTAAGATC ATCCTGTCAT                                                     500 TTATATGTAAAACTGCACTA TACTGGCCAT TATAAAAATT CGCGGCCGGG                                                     550 TGCGGTGGCTCATACCTGTA ATCCCAGCAC TTTGGGAGGC CGAAGCGGGT                                                     600 GGATCACTTGAGCCCTGGCG TTCGAGACCA GCCTGGGCAA CATGGTGAAA                                                     650 CCCCCGTCTCTACTAAAAAC ACAAAAACTA GCTGGGCGTG GTGGCAGGCG                                                     700 CCTGTAATCCCAGCTACTCA GGAGGCTGAG ACACGAGAAT CGCTTGAACC                                                     750 CGGGAGCAGAGGTTGCAGTG AGCCGAGATC ACGCCACTAG ACTCCATCCA                                                     800 GCCTGGGCGAAAGAGCAAGA CTCCGTCTCA AAAAAAAAAA TCGTTACAAT                                                     850 TTATGGTGGATTACTCCCCT CTTTTTACCT CATCAAGACA CAGCACTACT                                                     900 TTAAAGCAAAGTCAATGATT GAAACGCCTT TCTTTCCTAA TAAAAGGGAG                                                     950 ATTCAGTCCTTAAGATTAAT AATGTAGTAG TTACACTTGA TTAAAGCCAT                                                     1000 CCTCTGCTCAAGGAGAGGCT GGAGAAGGCA TTCTAAGGAG AAGGGGGCAG                                                     1050 GGTAGGAACTCGGACGCATC CCACTGAGCC GAGACAAGAT TCTGCTGTAG                                                     1100 TCAGTGCTGCCTGGGAATCT ATTTTCACAA AGTTCTCCAA AAAATGTGAT                                                     1150 GATCAAAACTAGGAATTAGT GTTCTGTGTC TTAGGCCCTA AAATCTTCCT                                                     1200 GTGAATTCCATTTTTAAGGT AGTCGAGGTG AACCGCGTCT GGTCTGCAGA                                                     1250 GGATAGAAAAAAGGCCCTCT CATACCTCAA GTTAGTTTCA CCTTTAAAGA                                                     1300 AGGTCGGAAGTAAAGACGCA AAGCCTTTCC CGGACGTGCG GAAGGGCAAC                                                     1350 GTCCTTCCTCATGGCCGGAA ATGGAACTTT AATTTCCCGT TCCCCCCAAC                                                     1400 CAGCCCGCCCGAGAGAGTGA CTCTCACGAG AGCCGCGAGA GTCAGCTTGG                                                     1450 CCAATCCGTGCGGTCGGCGG CCGCTCCCTT TATAAGCCGA CTCGCCCGGC                                                     1500 AGCGCACCGGGTTGCGGAGG GTGGGCCTGG GAGGGGTGGT GGCCATTTTT                                                     1550 TGTCTAACCCTAACTGAGAA GGGCGTAGGC GCCGTGCTTT TGCTCCCCGC                                                     1600 GCGCTGTTTTTCTCGCTGAC TTTCAGCGGG CGGAAAAGCC TCGGCCTGCC                                                     1650 GCCTTCCACCGTTCATTCTA GAGCAAACAA AAAATGTCAG CTGCTGGCCC                                                     1700 GTTCGCCCCTCCCGGGGACC TGCGGCGGGT CGCCTGCCCA GCCCCCGAAC                                                     1750 CCCGCCTGGAGGCCGCGGTC GGCCCGGGGC TTCTCCGGAG GCACCCACTG                                                     1800 CCACCGCGAAGAGTTGGGCT CTGTCAGCCG CGGGTCTCTC GGGGGCGAGG                                                     1850 GCGAGGTTCAGGCCTTTCAG GCCGCAGGAA GAGGAACGGA GCGAGTCCCC                                                     1900 GCGCGCGGCGCGATTCCCTG AGCTGTGGGA CGTGCACCCA GGACTCGGCT                                                     1950 CACACATGCAGTTCGCTTTC CTGTTGGTGG GGGGAACGCC GATCGTGCGC                                                     2000 ATCCGTCACCCCTCGCCGGC AGTGGGGGCT TGTGAACCCC CAAACCTGAC                                                     2050 TGACTGGGCCAGTGTGCTGC AAATTGGCAG GAGACGTGAA GGCACCTCCA                                                     2100 AAGTCGGCCAAAATGAATGG GCAGTGAGCC GGGGTTGCCT GGAGCCGTTC                                                     2150 CTGCGTGGGTTCTCCCGTCT TCCGCTTTTT GTTGCCTTTT ATGGTTGTAT                                                     2200 TACAACTTAGTTCCTGCTCT GCAGATTTTG TTGAGGTTTT TGCTTCTCCC                                                     2250 AAGGTAGATCTCGACCAGTC CCTCAACGGG GTGTGGGGAG AACAGTCATT                                                     2300 TTTTTTTGAGAGATCATTTA ACATTTAATG AATATTTAAT TAGAAGATCT                                                     2350 AAATGAACATTGGAAATTGT GTTCCTTTAA TGGTCATCGG TTTATGCCAG                                                     2400 AGGTTAGAAGTTTCTTTTTT GAAAAATTAG ACCTTGGCGA TGACCTTGAG                           2426 CAGTAGGATA TAACCCCCAC AAGCTT

The RNA component sequence begins at base 1459. A variety oftranscription control elements can also be identified in the sequence.An A/T Box consensus sequence is found at nucleotides 1438-1444; PSEconsensus sequences are found at nucleotides 1238-1250 as well asnucleotides 1406-1414; a CAAT box consensus sequence is found atnucleotides 1399-1406; an SP1 consensus sequence is found at nucleotides1354-1359; and a beta-interferon response element consensus sequence isfound at nucleotides 1234-1245.

The plasmids described above that were constructed during the cloning ofthe RNA component of human telomerase and the gene for the RNA componentare important aspects of the present invention. These plasmids can beused to produce the RNA component of, as well as the gene for, humantelomerase in substantially pure form, yet another important aspect ofthe present invention. In addition, those of skill in the art recognizethat a variety of other plasmids, as well as non-plasmid nucleic acidsin substantially pure form, that comprise all or at least a usefulportion of the nucleotide sequence of the RNA component of humantelomerase are useful materials provided by the present invention.

As a general point regarding the nucleic acids and preparationscontaining the same of the invention, those of skill in the artrecognize that the nucleic acids of the invention include both DNA andRNA molecules, as well as synthetic, non-naturally occurring analoguesof the same, and heteropolymers of deoxyribonucleotides,ribonucleotides, and/or analogues of either. The particular compositionof a nucleic acid or nucleic acid analogue of the invention will dependupon the purpose for which the material will be used and theenvironment(s) in which the material will be placed. Modified orsynthetic, non-naturally occurring nucleotides, have been designed toserve a variety of purposes and to remain stable in a variety ofenvironments, such as those in which nucleases are present, as is wellknown in the art. Modified or synthetic non-naturally occurringnucleotides, as compared to the naturally occurring ribo- ordeoxyribonucucleotides, may differ with respect to the carbohydrate(sugar), phosphate linkage, or base portions, of the nucleotide, or mayeven contain a non-nucleotide base (or no base at all) in some cases.See, e.g., Arnold et al., PCT patent Publication No. WO 89/02439,entitled “Non-nucleotide Linking Reagents for Nucleotide Probes”incorporated herein by reference.

Just as the nucleic acids of the invention can comprise a wide varietyof nucleotides, so too can those nucleic acids serve a wide variety ofuseful functions. One especially useful type of nucleic acid of theinvention is an antisense oligonucleotide that can be used in vivo or invitro to inhibit the activity of human telomerase. Antisenseoligonucleotides comprise a specific sequence of from about 10 to about25 to 200 or more (i.e., large enough to form a stable duplex but smallenough, depending on the mode of delivery, to administer in vivo, ifdesired) nucleotides complementary to a specific sequence of nucleotidesin the RNA component of human telomerase. The mechanism of action ofsuch oligonucleotides can involve binding of the RNA component either toprevent assembly of the functional ribonucleoprotein telomerase or toprevent the RNA component from serving as a template for telomeric DNAsynthesis.

Illustrative antisense oligonucleotides of the invention that serve toinhibit telomerase activity in vivo and/or in vitro include theoligonucleotides mentioned above in connection with the tests todetermine whether clone pGRN7 comprised the cDNA for the RNA componentof human telomerase. Three such oligonucleotides were synthesized as2′-O-methyl RNA oligonucleotides, which bind more tightly to RNA thanDNA oligonucleotides and are more resistant to hydrolysis thanunmodified RNA oligonucleotides, and, as noted above, were used todemonstrate inhibition of telomerase activity in vitro. The sequence ofeach of these O-methyl RNA oligonucleotides is shown below.

T3 5′-CUCAGUUAGGGUUAGACAAA-3′(SEQ ID NO:4)

P3 5′-CGCCCUUCUCAGUUAGGGUUAG-3′(SEQ ID NO:5)

TA3 5′-GGCGCCUACGCCCUUCUCAGUU-3′(SEQ ID NO:6)

These oligonucleotides can also be used to inhibit telomerase activityin human cells.

Those of skill in the art will recognize that the present inventionprovides a wide variety of antisense oligonucleotides able to inhibittelomerase activity. Another useful antisense oligonucleotides of theinvention is oligonucleotide Tel-AU, which has the sequence5′-CAGGCCCACCCTCCGCAACC-3′, (SEQ ID NO:7), and which, like any of theantisense oligonucleotides of the invention, can be synthesized usingphosphorothioate nucleotides, chiral-methyl phosphonates, naturallyoccurring nucleotides, or mixtures of the same to impart stability andthe desired T_(m). Those of skill in the art recognize that a widevariety of modified nucleotide analogues, such as O-methylribonucleotides, phosphorothioate nucleotides, and methyl phosphonatenucleotides, can be used to produce nucleic acids of the invention withmore desired properties (i.e., nuclease-resistant, tighter-binding,etc.) than those produced using naturally occurring nucleotides. Othertechniques for rendering oligonucleotides nuclease-resistant includethose described in PCT patent publication No. 94/12633.

Additional embodiments directed to modulation of telomerase activityinclude methods that employ specific antisense polynucleotidescomplementary to all or part of the human telomerase RNA component (hTR)sequences, such as antisense polynucleotides to the human hTR gene orits transcribed RNA, including truncated forms which may be associatedwith telomerase holoenzyme. Such complementary antisense polynucleotidesmay include nucleotide substitutions, additions, deletions, ortranspositions, so long as specific binding to the relevant targetsequence corresponding to hTR or its gene is retained as a functionalproperty of the polynucleotide. Complementary antisense polynucleotidesinclude soluble antisense RNA or DNA oligonucleotides which canhybridize specifically to hTR RNA species and prevent transcription ofthe hTR gene (Ching et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:10006; Broder et al. (1990) Ann. Int. Med. 113: 604; Loreau et al.(1990) FEBS Letters 274: 53; Holcenberg et al., WO91/11535; U.S. Ser.No. 07/530,165; WO91/09865;

WO91/04753; WO90/13641; and EP 386563, each of which is incorporatedherein by reference). The antisense polynucleotides therefore inhibitproduction of functional hTR. Since hTR expression (transcription rateand/or RNA stability) is associated with activation and enzymaticactivity of telomerase holoenzyme, antisense polynucleotides thatprevent transcription of RNA corresponding to hTR and/or the interactionof hTR to the protein component of human telomerase and/or theinteraction of hTR to telomeric sequences may inhibit telomeraseactivity and/or reverse a phenotype, such as immortalization orneoplastic transformation, of cells expressing telomerase activity inthe absence of antisense polynucelotides. Compositions containing atherapeutically effective dosage of hTR antisense polynucleotides may beadministered for treatment of diseases which require telomerase activityfor cellular pathogenesis (e.g., neoplasia) or to inhibit gameteproduction or maintenance (i.e., as a comntraceptive), if desired.Antisense polynucleotides of various lengths may be produced, althoughsuch antisense polynucleotides typically comprise a sequence of about atleast 25 consecutive nucleotides which are substantially complementaryto a naturally-occurring hTR polynucleotide sequence, and typicallywhich are perfectly complmentary to a human hTR sequence, often beingcomplementary to the sequence of hTR which is complemetary to thetelomere repeat sequence, or complementary to a portion of the hTR whichcontacts the telomerase polypeptide subunit.

Antisense polynucleotides may be produced from a heterologous expressioncassette in a transfectant cell or transgenic cell. Alternatively, theantisense polynucleotides may comprise soluble oligonucleotides that areadministered to the external milieu, either in the culture medium invitro or in interstitial spaces and bodily fluids (e.g., blood, CSF) forapplication in vivo. Soluble antisense polynucleotides present in theexternal milieu have been shown to gain access to the cytoplasm andinhibit specific RNA species. In some embodiments the antisensepolynucleotides comprise methylphosphonate moieties, C-5 propynylmoieties, 2′ fluororibose sugars, or are polyamide nucleic acids (PNAs)(Egholm et al. (1992) J. Am. Chem. Soc. 114: 1895; Wittung et al. (1994)Nature 368: 561; Egholm et al. (1993) Nature 365: 566; Hanvey et al.(1992) Science 258: 1481, incorporated herein by reference). For generalmethods relating to antisense polynucleotides, see Antisense RNA andDNA, (1988), D. A. Melton, Ed., Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.).

In addition to the antisense oligonucleotides of the invention, one canconstruct oligonucleotides that will bind to duplex nucleic acid eitherin the folded RNA component or in the gene for the RNA component,forming a triple helix-containing or triplex nucleic acid to inhibittelomerase activity. Such oligonucleotides of the invention areconstructed using the base-pairing rules of triple helix formation andthe nucleotide sequence of the RNA component (Cheng et al. (1988) J.Biol. Chem. 263: 15110; Ferrin and Camerini-Otero (1991) Science 354:1494; Ramdas et al. (1989) J. Biol. Chem. 264: 17395; Strobel et al.(1991) Science 254: 1639; Hsieh et al. (1990) op.cit.; Rigas et al.(1986) Proc. Natl. Acad. Sci. (U.S.A.) 83: 9591, incorporateed herein byreference). Such oligonucleotides can block telomerase activity in anumber of ways, including by preventing transcription of the telomerasegene or by binding to a duplex region of the RNA component of telomerasein a manner that prevents the RNA component either from forming afunctional ribonucleoprotein telomerase or from serving as a templatefor telomeric DNA synthesis. Typically, and depending on mode of action,the triplex-forming oligonucleotides of the invention comprise aspecific sequence of from about 10 to about 25 to 200 or more (i.e.,large enough to form a stable triple helix but small enough, dependingon the mode of delivery, to administer in vivo, if desired) nucleotides“complementary” (in this context, complementary means able to form astable triple helix) to a specific sequence in the RNA component oftelomerase or the gene for the RNA component of telomerase.

In addition to the antisense and triple helix-forming oligonucleotidesof the invention, “sense” oligonucleotides identical in sequence to atleast a portion of the RNA component of human telomerase can also beused to inhibit telomerase activity. oligonucleotides of the inventionof this type are characterized in comprising either (1) less than thecomplete sequence of the RNA component needed to form a functionaltelomerase enzyme or (2) the complete sequence of the RNA componentneeded to form a functional telomerase enzyme as well as a substitutionor insertion of one or more nucleotides that render the resulting RNAnon-functional. In both cases, inhibition of telomerase activity isobserved due to the “mutant” RNA component binding the proteincomponents of human telomerase to form an inactive telomerase molecule.The mechanism of action of such oligonucleotides thus involves theassembly of a non-functional ribonucleoprotein telomerase or theprevention of assembly of a functional ribonucleoprotein telomerase.Sense oligonucleotides of the invention of this type typically comprisea specific sequence of from about 20, 50 200, 400, 500, or morenucleotides identical to a specific sequence of nucleotides in the RNAcomponent of human telomerase.

Thus, another useful oligonucleotide of the invention comprises analtered or mutated sequence of the RNA component of human telomerase. Yuet al., 1990, Nature 344: 126, shows that a mutated form of the RNAcomponent of Tetrahymena telomerase can be incorporated into thetelomerase of Tetrahymena cells and that the incorporation hasdeleterious effects on those cells. Incorporation of mutated forms ofthe RNA component of human telomerase may have similar effects on humancells that otherwise have telomerase activity without affecting normalhuman cells that do not have telomerase activity. Such mutated formsinclude those in which the sequence 5′-CTAACCCTA-3′ (SEQ ID NO:8) ismutated to 5′-CAAACCCAA-3′ (SEQ ID NO:9), 5′-CTAACCCTA-3′ (SEQ IDNO:10), or 5′-CTCACCCTCA-3′ (SEQ ID NO:11). Each of these altered RNAcomponent sequences alters the telomeric repeat units incorporated intothe chromosomal DNA, thus affecting chromosome structure and function.Such oligonucleotides can be designed to contain restriction enzymerecognition sites useful in diagnostic methods for the presence of thealtered RNA component via restriction enzyme digestion of telomeric DNAor an extended telomerase substrate.

To illustrate this aspect of the invention, site-specific mutagenesiswas carried out using a plasmid (designated pGRN33, available from the.American Type Culture Collection under accession No ATCC 75925) thatcomprises an ^(˜)2.5 kb HindIII-SacI fragment from lambda clone 28-1(see Example 7, below) as well as the SV40 origin of replication (but nopromoter activity). The resulting plasmids, designated pGRN34(comprising 5′-CAAACCCAA-3′SEQ ID NO:9), pGRN36 (comprising5′-CCAACCCCAA-3′SEQ ID NO:10), and pGRN37 (comprising5′-CTCACCCTCA-3′SEQ ID NO:11), were transformed into eukaryotic hostcells (a 293-derived cell line expressing SV40 large T antigen), andtelomerase assays were conducted using cell extracts from thetransformants.

The assays showed that the telomerase activity in the cells resulted inthe formation of nucleic acids comprising the altered sequences,indicating that the genomic clone comprised a functional RNA componentgene and that the plasmids comprised an altered but functional RNAcomponent gene. These results illustrate how the present inventionprovides recombinant telomerase preparations and methods for producingsuch preparations. The present invention provides a recombinant humantelomerase that comprises the protein components of human telomerase infunctional association with a recombinant RNA component of theinvention. Such recombinant RNA component molecules of the inventioninclude those that differ from naturally occurring RNA componentmolecules by one or more base substitutions, deletions, or insertions,as well as RNA component molecules identical to a naturally occurringRNA component molecule that are produced in recombinant host cells. Themethod for producing such recombinant telomerase molecules comprisestransforming a eukaryotic host cell that expresses the proteincomponents of telomerase with a recombinant expression vector thatencodes an RNA component molecule of the invention, and culturing saidhost cells transformed with said vector under conditions such that theprotein components and RNA component are expressed and assemble to forman active telomerase molecule capable of adding sequences (notnecessarily the same sequence added by native telomerase) to telomeresof chromosomal DNA. Other useful embodiments of such recombinant DNAexpression vectors (or plasmids) include plasmids that comprise the genefor the RNA component of human telomerase with a deletion, insertion, orother modification that renders the gene non-functional. Such plasmidsare especially useful for human gene therapy to “knock-out” theendogenous RNA component gene, although a highly efficienttransformation and recombination system is required, to render thetreated cells irreversibly mortal.

Other oligonucleotides of the invention called “ribozymes” can also beused to inhibit telomerase activity. Unlike the antisense and otheroligonucleotides described above, which bind to an RNA, a DNA, or atelomerase protein component, a ribozyme not only binds but alsospecifically cleaves and thereby potentially inactivates a target RNA,such as the RNA component of human telomerase. Such a ribozyme cancomprise 5′- and 3′-terminal sequences complementary to the telomeraseRNA. Depending on the site of cleavage, a ribozyme can render thetelomerase enzyme inactive. See PCT patent publication No. 93/23572,supra. Those in the art upon review of the RNA sequence of the humantelomerase RNA component will note that several useful ribozyme targetsites are present and susceptible to cleavage by, for example, ahammerhead motif ribozyme. Illustrative ribozymes of the invention ofthis type include the ribozymes below, which are RNA molecules havingthe sequences indicated:

1: 5′-UAGGGUACUGAUGAGUCCGUGAGGACGAAACAAAAAAU-3′ (SEQ ID NO:12);

2: 5′-UUAGGGUCUGAUGAGUCCGUGAGGACGAAAGACAAAA-3′ (SEQ ID NO:13);

3: 5′-UCUCAGUCUGAUGAGUCCGUGAGGACGAAAGGGUUA-3′ (SEQ ID NO:14);

4: 5′-CCCGAGACUGAUGAGUCCGUGAGGACGAAACCCGCG-3′ (SEQ ID NO:15).

Other optimum target sites for ribozyme-mediated inhibition oftelomerase activity can be determined as described by Sullivan et al.,PCT patent publication No. 94/02595 and Draper et al., PCT patentpublication No. 93/23569, both incorporated herein by reference. Asdescribed by Hu et al., PCT patent publication No. 94/03596,incorporated herein by reference, antisense and ribozyme functions canbe combined in a single oligonucleotide. Moreover, ribozymes cancomprise one or more modified nucleotides or modified linkages betweennucleotides, as described above in conjunction with the description ofillustrative antisense oligonucleotides of the invention. In one aspect,a catalytic subunit of RNase P (human or E. coli) is modified (see,Altman S (1995) Biotechnology 13: 327) to generate a guide sequencewhich corresponds to the portion of hTR which basepairs to the telomererepeat sequence; such RNase P variants can cleave telomere sequences. Inone aspect, a catalytic subunit of RNase P (human or E. coli) ismodified to generate a guide sequence which is complementary to aportion of hTR such that the RNase P variant can cleave hTR RNA. Suchengineered ribozymes cen be expressed in cells or can be transferred bya variety of means (e.g., liposomes, immunoliposomes, biolisitics,direct uptake into cells, etc.). Other forms of ribozymes (group Iintron ribozymes (Cech T (1995) Biotechnology 13; 323); hammerheadribozymes (Edgington SM (1992) Biotechnology 10: 256) can be engineeredon the basis of the disclosed hTR sequence information to catalyzecleavage of hTR RNA and/or human telomere repeat sequences.

Thus, the invention provides a wide variety of oligonucleotides toinhibit telomerase activity. Such oligonucleotides can be used in thetherapeutic methods of the invention for treating disease, which methodscomprise administering to a patient a therapeutically effective dose ofa telomerase inhibitor or activator of the invention. One can measuretelomerase inhibition or activation to determine the amount of an agentthat should be delivered in a therapeutically effective dose using theassay protocols described in the copending U.S. patent applications andPCT patent publication No. 93/23572 noted above. As noted in thoseapplications and discussed above, inhibition of telomerase activityrenders an immortal cell mortal, while activation of telomerase activitycan increase the replicative lifespan of a cell. Telomerase inhibitiontherapy is an effective treatment against cancers involving theuncontrolled growth of immortal cells, and telomerase activation is aneffective treatment to prevent cell senescence. Delivery of agents thatinhibit or block telomerase activity, such as an antisenseoligonucleotide, a triple helix-forming oligonucleotide, a ribozyme, ora plasmid that drives expression of a mutant RNA component of telomerasecan prevent telomerase action and ultimately leads to cell senescenceand cell death of treated cells.

In addition, the present invention provides therapeutic methods thatensure that normal cells remain mortal; for instance, the RNA componentcan be modified using standard genetic engineering procedures to deleteall or a portion of a natural gene encoding the component (e.g., by invitro mutagenesis) by genetic recombination. Such cells will then beirreversibly mortal. This procedure is useful in gene therapy, wherenormal cells modified to contain expression plasmids are introduced intoa patient, and one wants to ensure cancerous cells are not introducedor, if such cells are introduced, then those cells have been renderedirreversibly mortal.

Because telomerase is active only in tumor, germline, and certain stemcells of the hematopoietic system, other normal cells are not affectedby telomerase inhibition therapy. Steps can also be taken to avoidcontact of the telomerase inhibitor with germline or stem cells,although this may not be essential. For instance, because germline cellsexpress telomerase activity, inhibition of telomerase may negativelyimpact spermatogenesis and sperm viability, suggesting that telomeraseinhibitors may be effective contraceptives or sterilization agents. Thiscontraceptive effect may not be desired, however, by a patient receivinga telomerase inhibitor of the invention for treatment of cancer. In suchcases, one can deliver a telomerase inhibitor of the invention in amanner that ensures the inhibitor will only be produced during theperiod of therapy, such that the negative impact on germline cells isonly transient.

Other therapeutic methods of the invention employ the telomerase RNAnucleic acid of the invention to stimulate telomerase activity and toextend replicative cell life span. These methods can be carried out bydelivering to a cell a functional recombinant telomeraseribonucleoprotein of the invention to the cell. For instance, theribonucleoprotein can be delivered to a cell in a liposome, or the genefor the RNA component of human telomerase (or a recombinant gene withdifferent regulatory elements) can be used in a eukaryotic expressionplasmid (with or without sequences coding for the expression of theprotein components of telomerase) to activate telomerase activity invarious normal human cells that otherwise lack detectable telomeraseactivity due to low levels of expression of the RNA component or aprotein component of telomerase. If the telomerase RNA component is notsufficient to stimulate telomerase activity, then the RNA component canbe transfected along with genes expressing the protein components oftelomerase to stimulate telomerase activity. Thus, the inventionprovides methods for treating a condition associated with the telomeraseactivity within a cell or group of cells by contacting the cell(s) witha therapeutically effective amount of an agent that alters telomeraseactivity in that cell.

Cells that incorporate extra copies of the telomerase RNA gene canexhibit an increase in telomerase activity and an associated extendedreplicative life span. Such therapy can be carried out ex vivo on cellsfor subsequent introduction into a host or can be carried out in vivo.The advantages of stabilizing or increasing telomere length by addingexogenous telomerase genes ex vivo to normal diploid cells include:telomere stabilization can arrest cellular senescence and allowpotentially unlimited amplification of the cells; and normal diploidcells with an extended life span can be cultured in vitro for drugtesting, virus manufacture, or other useful purposes. Moreover, ex vivoamplified stem cells of various types can be used in cell therapy forparticular diseases, as noted above.

Telomere stabilization can also suppress cancer incidence in replicatingcells by preventing telomeres from becoming critically short as cellsnear crisis. During crisis, massive genomic instability is generated asthe protective effect of the telomeric cap is lost. The “genetic deck”is reshuffled, and almost all cells die. The rare cells that emerge fromthis process are typically aneuploid with many gene rearrangements andend up reestablishing stability in their telomeres by expressingtelomerase. If crisis can be prevented by keeping telomeres long, thenthe genomic instability associated with crisis can also be prevented,limiting the chances that an individual cell will suffer the requirednumber of genetic mutations needed to spawn a metastatic cancer.

Cells that can be targeted for telomerase gene therapy (therapyinvolving increasing the telomerase activity of a target cell) includebut are not limited to hematopoietic stem cells (AIDS andpost-chemotherapy), vascular endothelial cells (cardiac and cerebralvascular disease), skin fibroblasts and basal skin keratinocytes (woundhealing and burns), chondrocytes (arthritis), brain astrocytes andmicroglial cells (Alzheimer's Disease), osteoblasts (osteoporosis),retinal cells (eye diseases), and pancreatic islet cells (Type Idiabetes).

Typically, the therapeutic methods of the invention involve theadministration of an oligonucleotide that functions to inhibit orstimulate telomerase activity under in vivo physiological conditions andwill be stable under those conditions. As noted above, modified nucleicacids may be useful in imparting such stability, as well as for ensuringdelivery of the oligonucleotide to the desired tissue, organ, or cell.Methods useful for delivery of oligonucleotides for therapeutic purposesare described in Inouye et al., U.S. Pat. No. 5,272,065, incorporatedherein by reference.

While oligonucleotides can be delivered directly as a drug in a suitablepharmaceutical formulation, one can also deliver oligonucleotides usinggene therapy and recombinant DNA expression plasmids of the invention.One such illustrative plasmid is described in Example 8, below. Ingeneral, such plasmids will comprise a promoter and, optionally, anenhancer (separate from any contained within the promoter sequences)that serve to drive transcription of an oligoribonucleotide, as well asother regulatory elements that provide for episomal maintenance orchromosomal integration and for high-level transcription, if desired.Adenovirus-based vectors are often used for gene therapy and aresuitable for use in conjunction with the reagents and methods of thepresent invention. See PCT patent publication Nos. 94/12650; 94/12649;and 94/12629. Useful promoters for such purposes include themetallothionein promoter, the constitutive adenovirus major latepromoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter,the MRP polIII promoter, the constitutive MPSV promoter, thetetracycline-inducible CMV promoter (such as the human immediate-earlyCMV promoter), and the constitutive CMV promoter. A plasmid useful forgene therapy can comprise other functional elements, such as selectablemarkers, identification regions, and other genes. Recombinant DNAexpression plasmids can also be used to prepare the oligonucleotides ofthe invention for delivery by means other than by gene therapy, althoughit may be more economical to make short oligonucleotides by in vitrochemical synthesis.

In related aspects, the invention features pharmaceutical compositionsincluding a therapeutically effective amount of a telomerase inhibitoror telomerase activator of the invention. Pharmaceutical compositions oftelomerase inhibitors of the invention include a mutant RNA component ofhuman telomerase, an antisense oligonucleotide or triple helix-formingoligonucleotide that binds the RNA component or the gene for the same ofhuman telomerase, or a ribozyme able to cleave the RNA component ofhuman telomerase, or combinations of the same or other pharmaceuticalsin a pharmaceutically acceptable carrier or salt. Other pharmaceuticalcompositions of the invention comprise a telomerase activatorpreparation, such as purified human telomerase or mRNA for the proteincomponents of telomerase and the RNA component of telomerase, and areused to treat senescence-related disease. In an aspect, a mutated sensehTR is administered to a cell population; said mutated sense hTRcomprises at least one base mismatch with respect to the humantelomerase repeat sequence, but is capable of exhibiting telomeraseactivity in conjunction with human telomerase polypeptide component,producing misincorporation at selected nucleotide positions in the humantelomerase repeat, thereby generating telomeres which rely on thecontinued presence of the mutated sense hTR for substantial replication.A therapeutic method is provided wherein a mutated sense hTR isadministered to a cell population for a sufficient period to introducetelomere sequences which are substantially non-functional as templatesfor naturally occurring hTR, followed by withdrawal of the mutated sensehTR which results in rapid loss of average telomere length in the cellpopulation and enhanced senescence or cell mortality.

The therapeutic agent can be provided in a formulation suitable forparenteral, nasal, oral, or other mode of administration. See PCT patentpublication No. 93/23572, supra.

Diagnostic Methods

The present invention provides diagnostic methods and reagents inaddition to the pharmaceutical formulations and therapeutic methodsdescribed above. The invention provides diagnostic methods fordetermining the level, amount, or presence of the RNA component of humantelomerase, telomerase, or telomerase activity in a cell, cellpopulation, or tissue sample. In a related aspect, the present inventionprovides useful reagents for such methods, optionally packaged into kitform together with instructions for using the kit to practice thediagnostic method. As noted above in connection with the tests conductedto determine that clone pGRN7 contained the cDNA for the RNA componentof human telomerase, the levels of the RNA component are elevated intumor cells. Thus, detection of the RNA component is a useful diagnosticfor tumor cells.

In addition, probes or primers that bind specifically to the RNAcomponent of human telomerase (or either strand of the gene for thesame) can be used in diagnostic methods to detect the presence oftelomerase nucleic acid in a sample. Primers and probes areoligonucleotides that are complementary, and so will bind, to a targetnucleic acid. Although primers and probes can differ in sequence andlength, the primary differentiating factor is one of function: primersserve to initiate DNA synthesis, as in PCR amplification, while probesare typically used only to bind to a target nucleic acid. Typicallengths for a primer or probe can range from 8 to 20 to 30 or morenucleotides. A primer or probe can also be labeled to facilitatedetection (i.e., radioactive or fluorescent molecules are typically usedfor this purpose) or purification/separation (i.e., biotin or avidin isoften used for this purpose).

An especially preferred diagnostic method of the invention involves thedetection of telomerase RNA component sequences in cell or tissuesamples taken from patients suspected to be at risk for cancer. Suchmethods will typically involve binding a labelled probe or primer to anRNA component sequence under conditions such that only perfectly matched(complementary) sequences bind (hybridize) to one another. Detection oflabelled material bound to RNA in the sample will correlate with thepresence of telomerase activity and the presence of cancer cells. Somecells may express the RNA component of telomerase but remaintelomerase-negative due to lack of expression of the protein componentsof telomerase. If one desired to detect the presence of telomeraseactivity in such cells, then one could first isolate protein and thendetermine whether the protein fraction contains the telomerase RNAcomponent, which would signal the presence of telomerase activity. Thediagnostic methods of the invention may be especially useful indetecting the presence of telomerase activity in tissue biopsies andhistological sections in which the method is carried out in situ,typically after amplification of telomerase RNA component using specificPCR primers of the invention.

Depending on the length and intended function of the primer, probe, orother nucleic acid comprising sequences from the RNA component of humantelomerase, expression plasmids of the invention may be useful. Forinstance, recombinant production of the full-length RNA component of theinvention can be carried out using a recombinant DNA expression plasmidof the invention that comprises a nucleic acid comprising the nucleotidesequence of the RNA component positioned for transcription under thecontrol of a suitable promoter. Host cells for such plasmids can beeither prokaryotic or eukaryotic, and the promoter, as well as the otherregulatory elements and selectable markers chosen for incorporation intothe expression plasmid will depend upon the host cell used forproduction.

The intact RNA component gene, i.e., the promoter, which includes anyregulatory sequences in the 5′-region of the gene, and RNA componentcoding region, can be used to express the RNA component in human cells,including human cells that have been immortalized by viraltransformation or cancer. The promoter of the RNA component gene may beregulated, however, and for this and other reasons, one may want toexpress the RNA component under the control of a different promoter. Onthe other hand, the promoter of the RNA component gene can be usedindependently of the RNA component coding sequence to express othercoding sequences of interest. For instance, one could study thetranscriptional regulation of the RNA component gene by fusing thepromoter of the RNA component gene to a coding sequence for a “reporter”coding sequence, such as the coding sequence for beta-galactosidase oranother enzyme or protein the expression of which can be readilymonitored. Thus, the promoter and other regulatory elements of the genefor the RNA component of human telomerase can be used not only toexpress the RNA component but also protein components of humantelomerase, antisense or other oligonucleotides, as well as other geneproducts of interest in human cells. Expression plasmids comprising theintact gene for the RNA component of human telomerase can be especiallyuseful for a variety of purposes, including gene therapy. Those of skillin the art recognize that a wide variety of expression plasmids can beused to produce useful nucleic acids of the invention and that the term“plasmid”, as used herein, refers to any type of nucleic acid (from aphage, virus, chromosome, etc.) that can be used to carry specificgenetic information into a host cell and maintain that information for aperiod of time.

As indicated by the foregoing description, access to purified nucleicacids comprising the sequence of the RNA component of human telomeraseprovides valuable diagnostic and therapeutic methods and reagents, aswell as other important benefits. One important benefit of the presentinvention is that the methods and reagents of the invention can be usedto isolate the RNA component and genes for the RNA component oftelomerase from any mammalian species that has an RNA componentsubstantially homologous to the human RNA component of the presentinvention. The phrase “substantially homologous” refers to that degreeof homology required for specific hybridization of an oligonucleotide ornucleic acid sequence of the human RNA component to a nucleic acidsequence of an RNA component sequence of another mammalian species.Given such substantial homology, those of ordinary skill in the art canuse the nucleic acids and oligonucleotide primers and probes of theinvention to identify and isolate substantially homologous sequences.

For instance, one can probe a genomic or cDNA library to detecthomologous sequences. One can also use primers corresponding to regionsof the RNA component sequence and PCR amplification under low ormoderate stringency conditions to amplify a specific homologous nucleicacid sequence from preparations of RNA or DNA from a mammalian species.By using these and other similar techniques, those of ordinary skill canreadily isolate not only variant RNA component nucleic acids from humancells but also homologous RNA component nucleic acids from othermammalian cells, such as cells from primates, from mammals of veterinaryinterest, i.e., cattle, sheep, horse, dogs, and cats, and from rodents,i.e., rats, mice, and hamsters. In turn, these nucleic acids can be usedto prepare transgenic animals of great value for screening and testingof pharmaceuticals that regulate telomerase activity. For instance, byusing a plasmid of the invention, one can “knock out” the RNA componentgene or replace the natural RNA component gene with a recombinantinducible gene in a mus spretus embryonic stem cell and then generate atransgenic mouse that will be useful as a model or test system for thestudy of age- or senescence-related disease. Example 9, below,illustrates how such methodology has been used to identify and isolateRNA component sequences of primates.

The reagents of the present invention also allow the cloning andisolation of nucleic acids encoding the protein components of human aswell as other mammalian telomerase enzymes, which have not previouslybeen available. Access to such nucleic acids provide complementarybenefits to those provided by the nucleic acids comprising nucleic acidsequences of the RNA component of human telomerase. For instance, and asnoted above, the therapeutic benefits of the present invention can beenhanced, in some instances, by use of purified preparations of theprotein components of human telomerase and by access to nucleic acidsencoding the same. The nucleic acids of the invention that encode theRNA component of human telomerase can be used to isolate the nucleicacid encoding the protein components of human telomerase, allowingaccess to such benefits. Thus, the invention provides methods forisolating and purifying the protein components of human telomerase, aswell as for identifying and isolating nucleic acids encoding the proteincomponents of human telomerase. In related aspects, the presentinvention provides purified human telomerase, purified nucleic acidsthat encode the protein components of human telomerase, recombinantexpression plasmids for the protein components of human telomerase. Theinvention also provides pharmaceutical compositions comprising as anactive ingredient either the protein components of human telomerase or anucleic acid that either encodes those protein components or interactswith nucleic acids that encode those protein components, such asantisense oligonucleotides, triple helix-froming oligonucleotides,ribozymes, or recombinant DNA expression plasmids for any of theforegoing.

The cloned RNA component of human telomerase can be used to identify andclone nucleic acids encoding the protein components of theribonucleoprotein telomerase enzyme. Several different methods can beemployed to achieve identification and cloning of the proteincomponents. For instance, one can use affinity capture of the enzyme orpartially denatured enzyme using as an affinity ligand either (1)nucleotide sequences complementary to the RNA component to bind to theRNA component of the intact enzyme; or (2) the RNA component to bind theprotein components of a partially or fully denatured enzyme. The ligandcan be affixed to a solid support or chemically modified (e.g.,biotinylated) for subsequent immobilization on the support. Exposure ofcell extracts containing human telomerase, followed by washing andelution of the telomerase enzyme bound to the support, provides a highlypurified preparation of the telomerase enzyme. The protein componentscan then be optionally purified further or directly analyzed by proteinsequencing. The protein sequence determined can be used to prepareprimers and probes for cloning the cDNA or identifying a clone in agenomic bank comprising nucleic acids that encode a protein component oftelomerase.

Affinity capture of telomerase utilizing an engineered RNA component canalso be conducted using in vitro transcribed telomerase RNA and a systemfor the reconstitution of telomerase enzyme activity. See Autexier andGreider, 1994, Genes & Development 8:563-575, incorporated herein byreference. The RNA is engineered to contain a tag, similar to epitopetagging of proteins. The tag can be an RNA sequence to which a tightlybinding ligand is available, e.g., an RNA sequence-specific antibody, asequence-specific nucleic acid binding protein, or an organic dye thatbinds tightly to a specific RNA sequence. The tolerance of telomerasefor the tag sequence and position can be tested using standard methods.Synthesis of the altered RNA component and the reconstitution step ofthis method can also be carried out in vivo. Affinity capture using theimmobilized ligand for the RNA tag can then be used to isolate theenzyme.

Expression screening can also be used to isolate the protein componentsof the telomerase enzyme. In this method, cDNA expression libraries canbe screened with labeled telomerase RNA, and cDNAs encoding proteinsthat bind specifically to telomerase RNA can be identified. A moleculargenetic approach using translational inhibition can also be used toisolate nucleic acids encoding the protein components of the telomeraseenzyme. In this method, telomerase RNA sequences will be fused upstreamof a selectable marker. When expressed in a suitable system, theselectable marker will be functional. When cDNA encoding a telomeraseRNA binding protein is expressed, the protein will bind to itsrecognition sequence thereby blocking translation of the selectablemarker, thus allowing for identification of the clone encoding theprotein. In other embodiments of this method, the blocked translation ofthe selectable marker will allow transformed cells to grow. Othersystems that can be employed include the “interaction trap system”described in PCT patent publication No. WO 94/10300; the “one-hybrid”system described in Li and Herskowitz, Dec. 17, 1993, Science262:1870-1874, and Zervos et al., Jan. 29, 1993, Cell 72:223-232; andthe “two-hybrid” system commercially available from Clontech.

Telomerase RNA binding or telomerase activity assays for detection ofspecific binding proteins and activity can be used to facilitate thepurification of the telomerase enzyme and the identification of nucleicacids that encode the protein components of the enzyme. For example,nucleic acids comprising RNA component sequences can be used as affinityreagents to isolate, identify, and purify peptides, proteins or othercompounds that bind specifically to a sequence contained within the RNAcomponent, such as the protein components of human telomerase. Severaldifferent formats are available, including gel shift, filter binding,footprinting, Northwestern (RNA probe of protein blot), andphotocrosslinking, to detect such binding and isolate the componentsthat bind specifically to the RNA component. These assays can be used toidentify binding proteins, to track purification of binding proteins, tocharacterize the RNA binding sites, to determine the molecular size ofbinding proteins, to label proteins for preparative isolation, and forsubsequent immunization of animals for antibody generation to obtainantibodies for use in isolating the protein or identifying a nucleicacid encoding the protein in a coupled transcription/translation system.

As will be apparent to those of skill in the art upon reading of thisdisclosure, the present invention provides valuable reagents relating tohuman telomerase, as well as a variety of useful therapeutic anddiagnostic methods. The above description of necessity provides alimited sample of such methods, which should not be construed aslimiting the scope of the invention. Other features and advantages ofthe invention will be apparent from the following examples and claims.

Oligonucleotides can be synthesized on an Applied Bio Systemsoligonucleotide synthesizer according to specifications provided by themanufacturer.

Methods for PCR amplification are described in the art (PCR Technology:Principles and Applications for DNA Amplification ed. H A Erlich,Freeman Press, New York, N.Y. (1992); PCR Protocols: A Guide to Methodsand Applications, eds. Innis, Gelf land, Snisky, and White, AcademicPress, San Diego, Calif. (1990); Mattila et al. (1991) Nucleic AcidsRes. 19: 4967; Eckert, K. A. and Kunkel, T. A. (1991) PCR Methods andApplications 1: 17; PCR, eds. McPherson, Quirkes, and Taylor, IRL Press,Oxford; and U.S. Pat. No. 4,683,202, which are incorporated herein byreference).

hTr polynucleotides and their complements may serve as hybridizationprobes or primers for detecting RNA or DNA sequences of hTR. For suchhybridization and PCR applications may contain substantial deletions,additions, nucleotide substitutions and/or transpositions, so long asspecific hybridization or specific amplification of a hTR sequence isretained. However, such nucleotide substitutions, deletions, andadditions should not substantially disrupt the ability of thepolynucleotide to hybridize to a hTR RNA or hTR gene sequence underhybridization conditions that are sufficiently stringent to result inspecific hybridization.

Specific hybridization is defined herein as the formation of hybridsbetween a probe polynucleotide (e.g., a polynucleotide of the inventionwhich may include substitutions, deletion, and/or additions) and aspecific target polynucleotide (e.g., a hTR RNA or hTR genomic genesequence, wherein the probe preferentially hybridizes to the specifictarget such that, for example, a single band corresponding to one ormore of the RNA species of the hTR gene (or specifically cleaved orprocessed hTR RNA species) can be identified on a Northern blot of RNAprepared from a suitable cell source (e.g., a somatic cell expressinghTR RNA). Polynucleotides of the invention which specifically hybridizeto hTR or human telomeric sequences may be prepared on the basis of thesequence data provided herein according to methods and thermodynamicprinciples known in the art and described in Maniatis et al., MolecularCloning: A Laboratory Manual, 2nd Ed., (1989), Cold Spring Harbor, N.Y.and Berger and Kimmel, Methods in Enzymology, Volume 152, Guide toMolecular Cloning Techniques (1987), Academic Press, Inc., San Diego,Calif., which are incorporated herein by reference.

Within the human population there can be minor alterations in the basicprimary sequence of hTR, including allelic variants, restriction sitepolymorphisms, and congenital hTR disease alleles associated withgenetic disease.

If desired, PCR amplimers for amplifying substantially full-length hTRcopies may be selected at the discretion of the practioner. Similarly,amplimers to amplify portions of the hTR gene or RNA may be selected.

Gene Therapy

Transferring exogenous genetic material into cells (i.e., DNA-mediatedtransfection) is an essential method for basic research in cell biologyand molecular genetics, as well as the basis for developing effectivemethods for human gene therapy. So far, the majority of the approvedgene transfer trials in the United States rely on replication-defectiveretroviral vectors harboring a therapeutic polynucleotide sequence aspart of the retroviral genome (Miller et al. (1990) Mol. Cell. Biol. 10:4239; Kolberg R (1992) J. NIH Res. 4: 43; Cornetta et al. (1991) Hum.Gene Ther. 2: 215). Adenoviral vectors have also been described forpotential use in human gene therapy (Rosenfeld et al. (1992) Cell 68:143).

The other gene transfer method that has been approved for use in humansis physical transfer of plasmid DNA in liposomes directly into tumorcells in situ. Unlike viral vectors which must be propagated in culturedcells, plasmid DNA can be purified to homogeneity and thus reduces thepotential for pathogenic contamination. In some situations (e.g., tumorcells) it may not be necessary for the exogenous DNA to stably integrateinto the transduced cell, since transient expression may suffice to killthe tumor cells. Liposome-mediated DNA transfer has been described byvarious investigators (Wang and Huang (1987) Biochem. Biophys. Res.Commun. 147: 980; Wang and Huang (1989) Biochemistry 28: 9508; Litzingerand Huang (1992) Biochem. Biophys. Acta 1113: 201; Gao and Huang (1991)Biochem. Biophys. Res. Commun. 179: 280; Felgner WO91/17424;WO91/16024).

Immunoliposomes have also been described as carriers of exogenouspolynucleotides (Wang and Huang (1987) Proc. Natl. Acad. Sci. (U.S.A.)84: 7851; Trubetskoy et al. (1992) Biochem. Biophys. Acta 1131: 311).Immunoliposomes hypothetically might be expected to have improved celltype specificity as compared to liposomes by virtue of the inclusion ofspecific antibodies which presumably bind to surface antigens onspecific cell types. Behr et al. (1989) Proc. Natl. Acad. Sci. (U.S.A.)86: 6982 report using lipopolyamine as a reagent to mediate transfectionitself, without the necessity of any additional phospholipid to formliposomes.

Accordingly, a polynucleotide substantially identical to at least 25nucleotides, preferably 50 to 100 nucelotides or more, of the hTRsequence or its complement, is operably linked to a heterologouspromoter to form a transcription unit capable of expressing an hTR RNAor an antisense hTR RNA in a human cell. Such a transcription unit maybe comprised in a transgene, adenoviral vector, or other gene therapymodality for delivery into human cells, sucha s for therapy of atelomerase-related disease (e.g., neoplasia). Suitable delivery methodsof the hTR sense or antisense expression construct will bne selected bypractitioners in view of acceptable practices and regulatoryrequirements.

The following examples describe specific aspects of the invention toillustrate the invention and provide a description of the methods usedto isolate and identify the RNA component of human telomerase for thoseof skill in the art. The examples should not be construed as limitingthe invention, as the examples merely provide specific methodologyuseful in understanding and practice of the invention.

EXAMPLE 1 Preparation of PCR-amplifiable cDNA

RNA was obtained from 293 cells by guanidine-thiocyanate extraction orfrom purified telomerase fractions by phenol/chloroform extractions. Thetotal RNA from 293 cells was size fractionated on a 2% agarose gel, andthe RNA below 500 bp was isolated.

First strand cDNA synthesis was performed with superscripts™ II reversetranscriptase obtained from Bethesda Research Laboratories (BRL). About0.5 to 1.0 μg RNA was mixed with about 40 ng of random primer (6 mer) inwater at a total volume of 11 μl. The solution was heated for 10 min. at95° C. and then cooled on ice for 5-10 min. The denatured nucleic acidwas collected by centrifugation. The denatured RNA and primer mixturewere then resuspended by adding, in the order shown: 4 μl 5×1st strandsynthesis buffer; 2 μl 0.1 M dithiothreitol (DTT); 1 μl RNAsin(Pharmacia); and 1 μl dNTP (0.125 mM each for 0.5 mM totalconcentration). The reaction mixture was incubated at 42° C. for 1 min.,and then, 1 μl (200 units) of Superscript™ II RTase (BRL) was added andmixed into the reaction, which was then incubated for 60 min. at 42° C.The resulting reaction mixture, containing the newly synthesized cDNAwas placed on ice until second strand synthesis was performed.

Second strand cDNA synthesis was performed as follows. About 20 μl ofthe reaction mixture from the first strand cDNA synthesis reactionmixture (from above) was mixed with, in the order shown, the followingcomponents: 111.1 μl of water; 16 μl of 10×E. coli DNA ligase buffer; 3μl of dNTP (2.5 mM each stock); 1.5 μl of E. coli DNA ligase (15 unitsfrom BRL); 7.7 μl of E. coli DNA polymerase (40 units from Pharmacia);and 0.7 μl of E. coli RNase H (BRL). The resulting solution was gentlymixed and incubated for 2 hours at 16° C., at which time 1 μl (10 units)of T4 DNA polymerase was added to the reaction tube and incubationcontinued for 5 min. at the same temperature (16° C.). The reaction wasstopped, and the nucleic acid was collected by extracting the reactionwith phenol/chloroform twice, precipitating the nucleic acid withethanol, and centrifuging the reaction mixture to pellet the nucleicacid.

The cDNA pellet collected by centrifugation was resuspended in 20 μl ofTE buffer and ligated to a double-stranded oligonucleotide called“NotAB” composed of two oligonucleotides (NH2 is an amino blockinggroup):

NotA: 5′-pATAGCGGCCGCAAGAATTCA-NH2 (SEQ ID NO:16)

NotB: 5′-TGAATTCTTGCGGCCGCTAT-3′ (SEQ ID NO:17)

The double-stranded oligonucleotide was made by mixing 50 μl of NotAoligonucleotide (100 pmol) with 50 μl of NotB oligonucleotide (100 pmol)in 46.25 μl of water, heating the resulting solution for 5 min. at 95°C., and adding 3.75 μl of 20×SSC buffer while the solution was stillhot. The tube containing the mixture was then placed in a beakercontaining hot water (at a temperature of about 70 to 75° C.), thetemperature of which was allowed to drop slowly to below 15° C., so thatthe two oligonucleotides could hybridize to form the double-strandedoligonucleotide NotAB. The resulting nucleic acid was collected byprecipitation and centrifugation.

The double-stranded NotAB oligonucleotide was resuspended in about 30 μlTE buffer and then ligated to the cDNA in a reaction mixture containing10 μl of the cDNA preparation described above, about 50 pmol (calculatedby OD260) of NotAB; 2 μl of 10×T4 DNA ligase buffer; 1.2 μl of T4 DNAligase; 0.2 μl of 10 mM ATP; and water in a total volume of 20 μl byincubating the reaction mixture at 16° C. overnight. The reaction wasthen heat-inactivated by heating the reaction mixture for 10 min. at 65°C. About 1 to 2 μl of the resulting mixture was typically used for PCRamplification; one can amplify the ligation mixture for 10 to 15 cycles(94° C., 45 seconds; 60° C., 45 seconds; and 72° C., 1.5 min.) and saveas a stock, as described in Example 2.

EXAMPLE 2 PCR Amplification of cDNA

The cDNA was routinely amplified by preparing an amplification reactionmixture composed of 5 μl of 10×PCR buffer (500 mM KCl; 100 mM Tris,pH=8.3; and 20 mM MgCl₂; 5-8 μl of dNTP (2.5 mM each); 1 μl of Taqpolymerase (Boehringer-Mannheim); 0.1 μl of gene 32 protein(Boehringer-Mannheim); 6 μl of Not B primer (20 μM stock); 2 μl of thecDNA (prepared as described in Example 1), and water to 50 μl . Thismixture was then overlayed with 50 to 100 μl of mineral oil, and PCRamplification was performed for 10 to 15 cycles of 94° C., 45 seconds;60° C., 45 seconds; and 72° C., 1.5 min. After amplification, thereaction mixture was extracted with phenol/chloroform, and the amplifiednucleic acid was precipitated with ethanol and collected bycentrifugation. The precipitate was then dissolved in 100 μl of TEbuffer to prepare a stock solution.

EXAMPLE 3 PCR Amplification for Cyclic Selection

To make PCR product for cyclic selection, about 1 μl of a stock solutionprepared as described in Example 2 was amplified in 50 μl of PCRreaction mixture prepared as described in Example 2, except that 21-24cycles of primer annealing, extension, and denaturation of product wereconducted. After amplification, reaction mixtures were extracted withphenol/chloroform, precipitated with ethanol, and collected bycentrifugation. Product yield was estimated by staining with ethidiumbromide after agarose gel electrophoresis of a small aliquot of thereaction mixture. Typically, about 2 μg of the nucleic acid product wereused for cyclic selection.

After cyclic selection, described in Example 4, about 1 to 2 μl of theselected “pull-down” products (out of a total volume of 20 μl ) were PCRamplified as described in Example 2 for 22 cycles, precipitated withethanol, and collected by centrifugation in preparation for furthercyclic selection.

EXAMPLE 4 Positive Selection of PCR-amplified cDNA

For the positive selection step of the cyclic selection process used toclone the RNA component of human telomerase, about 2 μg of thePCR-amplified cDNA were diluted into 25 μl of TE buffer and then mixedwith 1.25 μl of 20×SSC and the resulting solution heated to 95° C. for 3min. The temperature was lowered to 60° C. for 5 min., and one μl (0.1μg/μl ) of the R2 or R4 biotinylated probe was added. The sequences ofthese probes are shown below. The probes are O-methyl-RNA probes, so Uis O-methyl-uridine, A is O-methyl-riboadenine, G isO-methyl-riboguanine, and I is inosine.

R2: 5′-UUAGGGUUAGII-biotin (SEQ ID NO:18)

R4: 5′-AUUGGGUUAUII-biotin (SEQ ID NO:19)

The R2 probe is specific for the telomere repeat, and the R4 probe isspecific for RNase P, which was used to track the effectiveness andefficiency of the cyclic selection process. By carrying out a cyclicselection simultaneously but separately for RNase P RNA, a molecule ofknown sequence, one can have greater confidence that the cyclicselection process is functioning properly with respect to the moleculeof interest, in this case the RNA component of human telomerase. Aftereither the R2 or R4 probe was added to the mixture at 65° C., thetemperature of the hybridization reaction mixture was lowered to 30° C.by incubating the mixture at that temperature for 5 min., and then thereaction mixtures were further lowered to a temperature of 14° C. byincubating at that temperature for 60 min. Finally, the mixture wasincubated at 4° C. for 2-12 hours.

The entire hybridization reaction mixture for each sample (R2 or R4) wasadded to 400 μl of 0.5×SSC at 4° C. and then added to a tube of ice-coldmagnetic beads, which were purchased from Promega and pre-washed fourtimes with 0.5×SSC before use. The resulting mixture was incubated 30min. at 4° C. to ensure complete binding to the magnetic beads. Eachreaction tube was then incubated briefly at room temperature on themagnetic stand (Promega) to pull down the beads. The beads wereresuspended in cold 0.5×SSC (600 μl) and placed (in a tube) on ice. Thesamples were washed three more times with 0.5×SSC in this manner.Nucleic acid was eluted from the beads by resuspending the beads in 100μl of water and incubating for 2 min. at 65° C. before placing the beadsback on the magnetic stand for collection. This process was repeatedthree more times; the last time, the resuspended beads were incubatedfor 5 min. at 65° C. before placing the beads on the magnetic stand forcollection. All of the 100 μl supernatants (for each sample) were pooledand dried down to 20 μl in a SpeedVac™ centrifuge. The recovered DNA wasthen PCR amplified for another round of amplification and selection.After each amplification, the PCR products were phenol-chloroformextracted twice, ethanol precipitated, and resuspended in 20 μl of TEbuffer.

Typically, PCR amplifications were verified by agarose gelelectrophoresis. In addition, a variety of controls were used to monitorthe cyclic selection process. As one control, PCR “arms”(oligonucleotides of defined sequence that serve as primer hybridizationsites) were placed on a nucleic acid that comprised a neomycinresistance-conferring gene. The resulting nucleic acid was mixed withthe PCR-amplified cDNA and monitored at each selection by quantitativePCR. As another control, RNase P was followed in both the RNase Pselected and the telomerase RNA component selected libraries.

EXAMPLE 5 RT-PCR Protocol

The first strand cDNA was made in substantial accordance with theprocedure described in Example 1. Basically, RNA was purified from eachtelomerase fraction containing 0.1 to 1 μl RNA; typical, about one-thirdto one-fifth of the RNA made from a 300 μl fraction was used. The RNAwas mixed with 40 to 80 ng random hexamer in 10 μl , denatured for 10min. at 95° C. (using a thermal-cycling instrument), and chilled on ice.The denatured RNA and 6-mer were added to a reaction mixture containing4 μl of 5×1st strand synthesis buffer supplied by the manufacturer ofthe reverse transcriptase (RTase, purchased from BRL), 2 μl of 0.1 MDTT, 1 μl of 10 mM dNTP (each), 1 μl of RNase inhibitor (Pharmacia), andwater to a total volume of 9 μl . The combined mixture was placed into a42° C. water bath. After 1-2 min. incubation, 1 μl of Superscript™ IIRTase (BRL) was added to the mixture. The incubation was continued for60 min. at 42° C. The reaction was stopped by heating the tube for 10min. at 95-98° C. The first strand cDNA was collected by briefcentrifugation, aliquoted to new tubes, quickly frozen on dry ice, andstored at −80° C. or used immediately.

EXAMPLE 6 PCR Amplification of cDNA with a Specific Primer Set

For a 20 μl PCR reaction with radioactively labeled nucleotides, 1 μl ofthe cDNA prepared in accordance with the procedure of Example 5 wasmixed with 20 pmol of primer 1, 20 pmol of primer 2, 2.5 μl of 2.5 mMdNTP, 5 μCi of alpha-³²P-dATP, 2 units of Taq polymerase(Boehringer-Mannheim), 0.2 μg of T4 gene 32 protein(Boehringer-Mannheim), 2 μl of 10×buffer (500 mM KCl, 100 mMTris-HCl-pH8.3, and 20 mM MgCl₂), and water to a total volume of 20 μl.One drop of mineral oil was then added to the tube.

The PCR amplification conditions for the telomerase RNA component clonewere: 94° C. for 45 sec., 60° C. for 45 sec., 72° C. for 1.5 min. Thenumber of cycles differed depending on the type of purified materialsused for RNA preparation but typically range from 18 to 25 cycles. Asfor all quantitative RT-PCR, several reactions with differing cycleswere run for each sample to determine when the PCR amplification becamesaturated and non-linear.

For the RNase P used as a control, the PCR amplification conditionswere: 94° C. for 45 sec., 50° C. for 45 sec., and 72° C. for 1.5 min.Again, the number of cycles ranged from 15 to 22 cycles, depending onthe nature of the samples. The sequences of the primers used for RNase Pamplification are shown below:

P3: 5′-GGAAGGTCTGAGACTAG-3′ (SEQ ID NO:20)

P4: 5′-ATCTCCTGCCCAGTCTG-3′ (SEQ ID NO:21)

The PCR product obtained with these two primers is about 110 bp in size.

After PCR, the products (5 to 10 μl of the reaction mixture) were loadedonto a 6% native polyacrylamide gel and electrophoresed. Afterelectrophoresis, the gel was dried and exposed to a PhosphorImager™cassette or to autoradiographic film for analysis.

EXAMPLE 7 Cloning the Gene for the RNA Component of Human Telomerase

The procedures used to clone the gene for the RNA component of humantelomerase were carried out as generally described in Maniatis et al.,Laboratory Molecular Cloning Manual. A genomic DNA library of DNA fromthe human lung fibroblast cell line WI-38 inserted into phage lambdavector FIXII was purchased from Stratagene. The phage were plated at aconcentration of about 25,000 plaques per plate onto three sets of 15(150 mm) plates. The plates were made with NZY agar and NZY top agarose;the cells used for the phage transformation were XL1BlueMRAP2 cells; andthe transformants were grown overnight for about 16 hours at 37° C. Theplates were then chilled at 4° C. for about an hour, and then theplaques were “lifted” onto C/P nylon circles (filter paper from BioRad). This process was repeated to produce a duplicate set of liftedfilters. The filters (in duplicate) were denatured, neutralized,equilibrated in 6×SSC buffer, exposed to UV irradiation to cross-linkthe nucleic acid to the filter, and then dried on blotter paper.

Prehybridization was conducted for one hour at 37° C. in 50% formamidebuffer. The filters were probed with an ˜218 bp, radioactively-labeled,NotI fragment from clone pGRN7, which had been isolated byelectroelution from a 5% polyacrylamide gel after separation byelectrophoresis and then nick-translated with alpha-³²P-dCTP using anick-translation kit from Boehringer-Mannheim Biochemicals in accordancewith the manufacturer's instructions. About 25 ng (˜10 μCi label) of theprobe were used per filter, and hybridization was conducted overnight at37° C. in 50% formamide hybridization buffer. After hybridization, thefilters were washed at room temperature six times; the first threewashes were with 6×SSC containing 0.1% SDS, and the last three washeswere with 6×SSC alone. After an initial exposure of several duplicatefilters in a PhosphorImager™ cassette to check hybridization efficiencyand signal strength, the filters were washed at 65° C. in 0.5×SSC. Thefilters were then placed under Kodak XAR5 film using two intensifierscreens and allowed to expose the film for about 100 hours at −70° C.

One strong signal emanated from the filter containing a phage, laterdesignated 28-1, comprising the gene for the RNA component of humantelomerase. The plaque corresponding to the signal observed on thefilter was used to make secondary plates, so that an isolated plaque(confirmed by probing with labeled pGRN7 nucleic acid) could be culturedfor large-scale isolation of the phage DNA. Phage 28-1, available fromthe American Type Culture Collection under accession No. ATCC 75925,comprises an ^(˜)15 kb insert and comprises several restrictionfragments that contain sequences that hybridize with RNA componentsequences on pGRN7: a 4.2 kb EcoRI restriction enzyme fragment; a 4.2 kbClaI restriction enzyme fragment, and a 2.5 kb HindIII-SacI restrictionenzyme fragment. The latter fragment comprises the entire ^(˜)560nucleotide sequence of the RNA component shown above and is believed tocomprise the complete gene for the RNA component. The plasmid comprisingthe 2.5 kb HindIII-SacI restriction enzyme fragment in the pBluescriptvector was designated plasmid pGRN33 and is available from the AmericanType Culture Collection under the accession No. ATCC 75926. PlasmidpGRN33 was deposited under the Budapest Treaty of Oct. 25, 1994 at theAmerican Type Culture Collection, Rockville, Md. 20852. To the extentthe human gene may comprise sequences other than those on the 2.5 kbfragment, those sequences can be isolated from phage 28-1 or from otherphage clones identified by probing with the 2.5 kb fragment (or anotherprobe of the invention). The restriction enzyme fragments noted abovewere prepared in separate restriction enzyme digests; the products ofthe digests were separated by electrophoresis on a 0.7% agarose gel or,for the ^(˜)2.5 kb fragment only, a 3% polyacrylamide gel; and thedesired bands were cut from the gel and prepared for subcloning eitherby using the GeneClean™ Kit II (from Biol101, Inc.) or by electroelutioninto Spectropor #2 dialysis tubing in 0.1×TBE at 100 V for two hours(for the ^(˜)2.5 kb fragment only).

These restriction enzyme fragments were subcloned into E. coliexpression/mutagenesis plasmids derived from pUC-based plasmids or frompBluescriptII plasmids that also comprise an SV40 origin of replication(but no SV40 promoter activity). The resulting plasmids can be used toprepare altered (mutated) RNA component nucleic acids for introductioninto human or other eukaryotic cells for a variety of purposes, asdescribed above in the Description of the Preferred Embodiments.

EXAMPLE 8 Antisense Plasmids for the RNA Component of Human Telomerase

Antisense expression plasmids were prepared by PCR amplification of RNAcomponent cDNA using the following primer sets: (1) NotB and G1, whichproduces an antisense nucleic acid that is smaller than the cDNA insertin the plasmid; and (2) NotB and R3C, which produces a full-length(relative to the insert in the plasmid) antisense nucleic acid. Thenucleotide sequence of NotB is shown in Example 1, above; the nucleotidesequences of the G1 and R3C primers are shown below.

G1: 5′-GAGAAAAACAGCGCGCGGGGAGCAAAAGCA-3′ (SEQ ID NO:22)

R3C: 5′-GTTTGCTCTAGAATGAACGGTGGAAG-3′ (SEQ ID NO:23)

After PCR amplification, the amplified fragments were cloned into an^(˜)10 kb expression plasmid at a PmlI site; the plasmid comprisespuromycin resistance-conferring, DHFR, and hygromycin Bresistance-conferring genes as selectable markers, the SV40 origin ofreplication; the inducible human metallothionein gene promoterpositioned for expression of the antisense strand of the gene for theRNA component of human telomerase (one could also use a strongerpromoter to get higher expression levels), and the SV40 late poly Aaddition site.

The resulting plasmids (designated pGRN42 for the NotB/G1product andpGRN45 for the NotB/R3C product) were transfected by the calciumphosphate procedure (see Maniatis et al., supra) into the fibrosarcomacell line HT1080. HT1080 cells are normally immortal; expression of theantisense RNA for the RNA component of human telomerase should preventthe RNA component of human telomerase from association with the proteincomponents, blocking the formation of active telomerase and renderingthe cells mortal.

EXAMPLE 9 Identification and Isolation of RNA Component Nucleic Acidsfrom Non-human Mammals

To illustrate how the reagents of the invention can be used to identifyand isolate substantially homologous nucleic acids from other mammalianspecies, PCR primers complementary to human RNA component sequences wereused to amplify homologous sequences in a PCR. An illustrative primerpair used to demonstrate this aspect of the invention is composed ofprimer +10, which has the sequence 5′-CACCGGGTTGCGGAGGGAGG-3′ (SEQ IDNO:24), and primer R7, which has the sequence5′-GGAGGGGCGAACGGGCCAGCA-3′ (SEQ ID NO:25). Genomic DNA was preparedfrom chimpanzee, squirrel monkey, rhesus monkey, and baboon tissue anddissolved in TE buffer at a concentration of about 0.5 -4 mg/ml.

For each tissue type, a PCR mixture was prepared, which mixturecomprised: 1 μL of genomic DNA, 48 μL of Master Mix (Master Mix iscomposed of 1× TaqExtender™ buffer from Stratagene, 200 μM of each dNTP,and 0.5 μM of each primer), and 0.5 μL of a 1:1 mixture of Taqpolymerase (5 Units/μL, Boehringer Mannheim):Tth polymerase(TaqExtender™ polymerase, from Stratagene). The reaction tubes wereloaded onto a thermal cycler, which was programmed to first heat thereaction mixture at 94° C. for 5 minutes and then to perform 27 cyclesof incubations at 94° C. for 30 sec., 63° C. for 10 sec., and 72° C. for45 sec. After the amplification reaction was complete, about 10 AL ofeach reaction mixture were loaded onto a 2% agarose gel forelectrophoresis. After electrophoresis, staining of the gel, and UVirradiation, one could observe that each reaction mixture contained aband of the predicted (^(˜)200 bp) size. Nucleic acids from these bandscan be cloned and sequenced and the remainder of the RNA component genesfrom each of these mammalian species can be cloned as described abovefor the gene for the RNA component of human telomerase.

EXAMPLE 10 Mutated Sense hTR Sequences

This example demostrates that changing the hTR sequence at the templateregion alters the sequence synthesized by human telomerase, leading tochanges in the chromosomal telomerase repeats.

To determine if re-programming the TRC3 template region would produceleads to corresponding changes in the telomere repeat synthesized inhuman telomerase activity, a gene fragment that expresses the entire hTR(TRC3) gene sequence was cloned and mutagenized. Southern blot analysisshowed that TRC3 hybridized to a single-copy gene in the human genome. Agenomic copy of TRC3 was isolated from a lambda phage library and shownto have the same restriction map as that observed on genomic Southernblots probed with TRC3. A 2.6 kb HindIII-Sac1 fragment was isolated andsubcloned into a modified version of pBluescript, generating the genomicTRC3 plasmid pGRN33. The 5′ and 3′ ends of the RNA were mapped usingprimer extension, RACE PCR, and RT-PCR. The size of the RNA transcriptwas ≈550 bases, consistent with its migration on Northern blots. Thetranscribed region for TRC3 was central to the genomic clone with 1.4 kbof upstream sequence.

RNA was extracted from purified telomerase preparations and was thenused in the following experiments. 5′ RACE: First strand cDNA was madeusing antisense TRC3 primers (R3B: 5′-CAACGAACGGCCA GCAGCTGACATT; SEQ IDNO:27) in the presence of ³²P-DATP. Extension products were resolved byPAGE and were identified by autoradiography, excised and extracted. Anoligonucleotide (NotA: 5′-pATAGCGGCCGCTT GCCTTCA; SEQ ID NO:28) wasligated to these first strand products using T4 RNA ligase and PCR wasthen performed using a nested primer, R3c(5′-GTTTGCTCTAGAATGAACGGTGGAAG; SEQ ID NO:23) and an oligonucleotide(NotB: 5′-TGAATTCTTGCGGCCGCTAT; SEQ ID NO:17) complementary to the NotAoligonucleotide. The PCR products were resolved on an agarose gel andbands were excised and sequenced directly, using oligonucleotide G1(5′-AGAAAAACAGCGCGCGGGGAGCAAAGCA; SEQ ID NO:29) as a primer. 3′ endmapping: Attempts to perform 3′ RACE were unsuccessful. Subsequently, anRT-PCR strategy was employed in which a series of antisense primerscomplementary to the genomic DNA sequence were used to prime firststrand cDNA synthesis. The primers in this series were spaced atapproximately 150 bp intervals starting from the 5′ end of thetranscript, proceeding towards the 3′ end. PCR was then performed usingthe first strand primer and a primer whose sequence was internal to theknown TRC3 transcript (F3b: 5′-TCTAACCCTAACTGAGAAGGGCGTAG; SEQ IDNO:30). Reverse transcriptase-sensitive PCR bands of the predicted sizewere seen when using cDNA made with all primers designed to the interval+100 to +450 (numbering relative to 5′ end) but not with any of theprimers designed to +558 to +950. This placed the putative 3′ end of themature TRC3 transcript between position +450 and +558. Subsequent roundsof primer design and RT-PCR narrowed this interval to between +545 and+558.

The predicted template sequence of TRC3 was altered from CUAACCCUA (SEQID NO:31) to CCAACCCCA (SEQ ID NO:32) (MuC) or CAAACCCAA (SEQ ID NO:9)(MuA) by in vitro mutagenesis (performed substantially according toPerez et al. (1994) J. Biol. Chem. 269: 22485) of the genomic plasmidpGRN33. If incorporated into functional telomerase, these mutant RNAsshould template the synthesis of TTGGGG (MuC) or TTTGGG (MuA) ratherthan wild-type repeat TTAGGG. These mutant telomerase activities couldbe easily distinguished from wild-type activity since they would nolonger require DATP for activity and only the wild-type activity shouldbe sensitive to termination by ddATP. A double mutant (MuC+17) was alsoprepared in which a 17 bp insertion was present at +180 bp in additionto the MuC template. This mutant allowed specific detection of thealtered RNA with a probe to the 17 bp insertion or by size.

The 2.6 kb of genomic sequence was sufficient for expression of TRC3 invivo. Cells were transiently transfected with the MuC+17 plasmid and RNAexpressed from the transfected DNA was detected by RT PCR using the 17bp insert sequence as the primer in the reverse transcription step. TheRNA was detected in MuC+17-transfected cells but not in mock transfectedcells, indicating that the 2.6 kb genomic clone was sufficient for TRC3expression. Stable transformants were then derived from each of thesethree mutant plasmids by electroporation of HT1080 cells along withpCMVneo. Resistant clones were selected in G418 and expression of theMuC+17 RNA was verified by RT-PCR (Irving et al. (1992) Exp. Cell Res.202: 161).

To test for mutant telomerase activity, the extracts were assayed fromuntransfected cells and from three stable transformants with integratedMuC*, MuC, or MuA vectors (C*, C, or A in FIG. 1). Since the mutantextracts were expected to contain both wild-type and mutant telomeraseactivities, various assay conditions were employed to distinguishbetween them. Under normal reaction conditions (dTTP, ³²P-dGTP andDATP), all three extracts from the mutant construct series showedwild-type telomerase activity that was sensitive to RNase (FIG. 1, lanes1-6). As expected, this activity was unaffected when ddCTP was includedin the reactions (FIG. 1, lanes 7-9) and was abolished by ddTTP (lanes10-12). In contrast, when ddATP was substituted for DATP, the C (lane14) and A (lane 15) extracts still displayed RNase-sensitive (lanes 17and 18) telomerase activity, while C* (lane 13) and control extract didnot. These results indicate that the ddATP-resistant activitiesrepresent telomerase was reprogrammed with MuC or MuA TRC3 RNA. Incontrast, the 17 bp insertion in C* inhibits reconstitution, indicatingthat telomerase reconstitution is very specific for the TRC3 sequence.

To confirm that the sequence synthesized by the mutant MuA was(GGGTTT)n, we modified the existing PCR methodology for amplifyingtelomerase repeats added onto a unique telomerase primer (Kim et al.(1994) Science 266: 2011). Using synthetic primers, we identifiedreaction conditions where a 3′ primer with the sequenced(CCCAAACCCAAACCCAA) (SEQ ID NO:33) would only amplify (TTTGGG)n repeatsand would not amplify (TTAGGG)n containing repeats.

To distinguish between telomeric repeats added by wild-type versusmutated telomerase, a two-step assay was combined with a strategy forlimiting the availability of nucleotides for the telomerase reaction.Since MuA and MuC would generate telomeric repeat sequences of(TTTGGG)_(n) and (TTGGGG)_(n), respectively, cell extracts were firstincubated with the TS substrate in the presence of only dTTP and dGTPfor 10 min at room temperature to allow for the addition of telomericrepeats. Residual telomerase activity was then destroyed by boiling theextracts for 5 min. Telomerase products with specific DNA sequence werethen detected by PCR amplification using the appropriate reverse primersand in the presence of all 4 dNTP's and trace amounts of ³²P-dCTP asdescribed (Kim et al. (1994) op.cit). To detect the MuA products, thereverse primer was (ACCCAA)₄ (SEQ ID NO:34) and the PCR conditions were94° C., 10 sec, 60° C., 30 sec and 72° C., 30 sec for 20 cycles. Todetect the MuC products, three reverse primers were used: (CCCCAA)₃,(SEQ ID NO:35) (CCAACC)₃ and (CCAACC)₃ (SEQ ID NO:36), respectively,which gave PCR products with the corresponding mobility shiftsconsistent with the expected position of annealing to the telomeraseproducts. The PCR conditions used were the same as above, except thatthe annealing temperature was 50° C. Under the same conditions, notelomerase products were generated from extracts of parental cells orcells transfected with the wild-type TRC-3 gene. In tests of thespecificity of our PCR amplification conditions, syntheticoligonucleotides containing (TTTGGG)_(n) and (TTGGGG)_(n) generated theappropriate 6 nt ladder PCR products with (ACCCAA)₄ (SEQ ID NO:34) or(CCCCAA)₃ (SEQ ID NO:35) reverse primer respectively, whereas(TTAGGG)_(n) oligonucleotides did not prodce any PCR products with the(ACCCAA)₄ (SEQ ID NO:34) or (CCCCAA)₃ (SEQ ID NO:35) reverse primer.

Using these conditions, extracts from MuA but not from MuC or wild typecells generated products in the modified telomerase assay, indicatingthat telomerase from MuA containing cells generated (TTTGGG)n repeats.Similar methods were used to analyze the MuC mutant, which synthesized(TTGGGG)n repeats. Together, the above data constitute strong evidencethat the TRC3 gene encodes the RNA component of human telomerase, andthus we have renamed TRC3 as hTR for human Telomerase RNA.

EXAMPLE 11 Expression of hTR in Mortal and Immortal Cells

Most cancer cells express high levels of telomerase activity, while inmost normal somatic human cells, telomerase is not detected (Kim et al.(1994) op.cit). To determine if hTR RNA levels are elevated in immortalcancer cell lines, hTR and GAPDH transcript levels were analyzed usingRT-PCR in five mortal primary cell strains, which lacked detectabletelomerase activity, and five immortal cancer cell lines with highlevels of telomerase activity. The steady state levels of hTRtranscripts was 3- to 12-fold higher in the tumor cell lines than inprimary cells when compared to the levels of GAPDH (FIG. 2A). While hTRlevels were increased in the immortal cancer cells expressing highlevels of telomerase, it is interesting that low but readily detectablelevels of hTR RNA were also present in mortal primary cells with nodetectable telomerase activity (lanes 1-5).

The hTR levels were also examined in a variety of normal human tissuesby Northern blot analysis. Testes and ovary had the highest level ofhTR, which was expected since these tissues express high levels oftelomerase activity. However, a number of other tissues also expressedhTR (FIGS. 2B and 2C). These include normal kidney, prostate, and adultliver, all of which lack detectable levels of telomerase activity. Theseresults confirm the data from cell lines (FIG. 2A) and suggest thattelomerase RNA may be present but inactive in a number of human tissues.Similar tissue-specific differences in RNA expression are seen in mousetissues; however, many normal mouse tissues are positive for telomeraseactivity. The apparent increased repression of telomerase activity inhuman cells may help explain why mouse cells spontaneously immortalizein culture while human cells do not.

EXAMPLE 12 Expression of Antisense hTR (Human Telomerase RNA Component)Transcripts in HeLa Cells Leads to Cell Crisis and Cell Death

To examine the function of telomerase in an immortalized cell, antisensehTR expression constructs were introduced into.HeLa cells. A 200 bpEcoRI DNA fragment containing 1 to 193 bp of a human telomerase RNAcomponent cDNA clone, TRC3, was inserted into the EcoRI site of p10-3and pBBS212 to generate plasmids p10-3-hTR and pBBBhTR, respectively.Plasmid p10-3-hTR expresses the antisense of hTR under thetranscriptional control of the tetracycline-regulated CMV minimalpromoter relative to the five Tet-operators upstream in two differentorientations as described (Gossen et al. (1992) Proc. Natl. Acad. Sci.(USA) 89: 5547). Plasmid pBBS-hTR expresses the antisense of hTR underthe control of the MPSV promoter (Lin et al. (1994) Gene 147: 287). Inparallel, control expression vectors lacking the antisense hTR codingsequence were also electroporated into HeLa cells. Clones containingantisense or control plasmids were selected in three separateexperiments. Initially, the 41 cultures expressing antisense hTR grewidentically to cells with the control vector. However, at 23 to 26population doubling levels (PDL) post-transfection, 33 out of 41antisense expressing cultures underwent crisis (Table I). Cell crisis inthese cultures was characterized by a marked inhibition in cell growthfrom 20 to 26 PD, followed by the rounding up and detachment of cellsfrom the plate over a period of a week. In 28 out of 33 cases in whichthe cells underwent crisis, rare (<1%) revertant colonies were observedwithin three weeks after most of the cells had died. The revertant cellsmay represent variants that escape the inhibiting effect of theantisense hTR construct. In contrast to the antisense clones, none ofthe vector control cell lines had any change in growth or mortality over50 doublings.

TABLE I Antisense hTR leads to shorter mean TRF and cell crisis Threeindependent experiments were carried out in which HeTe7 cells (HeLacells that express high levels of the tetracycline repressor- VP16chimeric protein) were transfected by electroporation with eitherplasmid p10-3-hTR, expressing antisense hTR under control of thetetracycline-VP16-induced CMV minimal promoter or pBBS-hTR, expressingantisense hTR under the control of the MPSV promoter (54). [In theseexperiments no regulation by tetracycline was observed. The experimentswith p10-3-hTR were typically carried out in the absence oftetracycline, because control experiments with luciferase or antisensehTR under the control of the tetracycline- VP16-induced CMV minimalpromoter demonstrated that the presence or absence of tetracycline hadlittle effect on luciferase or antisense hTR expression in HeTe7 cells.Indeed, in early experiments with antisense hTR constructs in HeTe7cells, the cells still underwent crisis at 23 to 26 PDL in the presenceof tetracycline.]. As a control, HeTe7 cells were also transfected withthe parental vector under the same conditions. Eleven to eighteen stableclones were isolated from each transfection series. Clones from allisolates displayed identical morphology and growth profiles until 20PDL, at which time the growth of most of the antisense-expressing clonesslowed markedly (p10-3-hTR and PBBS-hTR). By PDL 23-26, these cellsunderwent crisis, characterized by the appearance of enlarged androunded cells. However, not all of the clones generated from thepBBS-hTR transfected HeTe7 cells underwent crisis; eight clonesexpressing antisense hTR continued growing similar to the controlcultures. Cells from all clones were harvested at PDL 23 and the meanTRF lengths were determined. The P values were calculated by the methodof unpaired t-test. For each series, the proportion of clones thatunderwent crisis is indicated. Plasmid Cell Crisis Mean TRF P ValueCrisis/Total 1 p10-3 no ND 0/7 p10-3-hTR yes ND 18/18 2 p10-3 no 3.27 ±0.10  0/12 p10-3-hTR yes 2.72 ± 0.07 0.0003 11/11 3 pBBS no 3.22 ± 0.11 0/13 pBBS-hTRa yes 2.39 ± 0.10 0.0008 4/4 pBBS-hTRb na 3.03 ± 0.200.3551 0/8 HeTe7 cells no 3.15 ± 0.09 Parental cells

To determine if telomere length and telomerase inhibition correlatedwith cell crisis in the antisense expressing clones, telomere length andtelomerase activity in several precrisis control and experimentalcolonies were assayed at 23 PDL. All colonies containing control vectorhad mean TRF lengths (3.22 and 3.27 kb) similar to the parent cell line(3.15 kb), whereas clones containing the antisense vector constructsthat underwent crisis had mean TRF lengths between 2.39 and 2.72 kb or17 to 26% shorter than those of the parental line (FIG. 3). These dataare consistent with telomere repeats being lost in the antisensecontaining clones due to the inhibition of telomerase activity. To testthis directly, telomerase activity was assayed in 14 of the clones.Telomerase activity was generally low but detectable in many of theantisense clones, although the shortened telomeres suggest that thelevel was not sufficient to maintain telomere length since mean TRF fellfrom 3.22 to 2.39 kb (P=0.0008). In the eight clones containing theantisense vector (pBBS-hTRb) that did not undergo crisis, telomerelength was not significantly changed (3.03 versus 3.33, P=0.355), andtelomerase activity was similar to that of controls. Taken together,these results are evidence that telomere loss leads to crisis and celldeath once telomeres reach a critical length.

The induction of cell crisis in HeLa cells expressing antisense hTRprovide further support that telomerase inhibition can provide aspecific and effective therapeutic against human cancer.

EXAMPLE 13 In Situ Amplification and Detection In Situ Detection ofTelomerase RNA

A. Fluorescent in Situ Hybridization (FISH)

Identification of telomerase positive cells in a mixed population ofcells or tissues can be peformed by in situ hybridization of labeledprobe targetted at the RNA component of telomerase. A tissue of cellsamples fixed onto a microscopic glass slide and permeabilizedsufficiently used for in situ hybridization. An example of in situhybridization for human telomerase RNA (htRNA) consist of firstdenaturing the nucleic acids by immersing the slides in 70% deionizedformamide/ 2×SSC solution pre-warmed to 70-74° C. for 2-3 min. Theslides are then transferred to ice-cold 70% EtOH, and then to 95% EtOH,and then to 100% EtOH (4 min. in each solutions). 100-200 ng (per slide)of the labeled htRNA probe (˜500 bp DNA probe labeled with biotin,digoxigenin, radioisotope, fluorescent tag) is dried, resuspended in 10μl of 100% deionized formamide, denatured by incubation at 75° C. for 8min., and immediately cooled on ice. Add 10 μl of 2×hybridization buffer(4×SSC; 4×Denhardt's solution; 20% dextran sulfate; 100 mM Tris, pH 7.5)to the 10 μl of resuspended probe. Add the probe/hybridization mix (20μl) to the fixed sample, overlay with a coverslip, and seal thecoverslip with rubber cement or nail polish. Incubate the sample at 37°C. for 8-48 hr. After the hybridization, remove the coverslip, wash thesample twice in 2×SSC/50% deionized formamide at 37° C., and then washtwice in 2×SSC at 37° C. (5 min. per wash). The sample is then viewedunder a microscope.

B. Primed-in Situ Labeling (PRINS)

Another variation to the traditional in situ hybridization detectionmethod is Primed-in situ labeling (PRINS). Detection of the htRNA byPRINS consist of initial cDNA synthesis of the htRNA by reversetranscriptase (RT) followed by PRINS detection using htRNA-specificoligonucleotide probe and chain elongation incorporating labelednucleotides. RT reaction of the htRNA can be performed by variety ofmethods. One example of the RT reaction is by using GeneAmp ThermostablerTth Reverse Transcriptase RNA PCR kit (Perkin Elmer). In this method,10 μl of RT mix (10 mM Tris-HCl pH 8.3; 90 mM KCl; 1 mM MnCl₂; 200 μMdNTPs; 2.5 U rTth DNA polymerase; 0.4 μM return primer [e.g., R7G:5′-GGAGGGGCGAACGGGCCAGCAG-3′]) is placed on the fixed and permeabilizedsample (Sec. I.B), sealed with coverslip, anchored with nail polish,overlayed with mineral, and incubated at 70° C. for 30 min. Afterwards,the mineral oil is removed by washing for 5 min in xylene, and then 5min in 100% EtOH. The coverslip is taken off, washed briefly with DepCwater, then with 100% EtOH, and air dried for 5 min. Then 10 μl of PRINSmixture (5% (v/v) glycerol; 10 mM Tris-HCl, pH 0.3; 100 mM KCl; 0.05%(w/v) Tween 20; 0.75 mM EGTA; 2.5 mM MgCl₂; 0.4 μM forward primer [e.g.U3b: 5′-GCCTGGGAGGGGTGGTGGCTATTTTTTG-3′]; 200 μM dA, dG, dCTP; 110 μMdTTP; 90 μM labeled dUTP) is placed on the sample. Sealed withcoverslip, anchored with nail polish, overlayed with mineral oil, andincubated at 70° C. for 30 min to 3 hr. As soon as the last PCR step iscompleted, the sample is washed 3 times in the wash buffer (4×SSC; 0.05%Tween 20) heated to 70° C., for 2 min. The signal is then observed.

C. In Situ RT-PCR

RT-PCR detection of htRNA consist of cDNA synthesis of the target RNA byreverse transcriptase reaction (viral reverse transcriptase, or by theintrinsic RT activity of thermostable DNA polymerases), followed by insitu PCR amplification of the target cDNA. Various RT reactions can beused in the cDNA synthesis of the htRNA including the RT protocoldiscussed in Sec. II.B. Furthermore, the same buffer condition and theprimers used in the PRINS detection methods can also be used for RT-PCR,only difference being that instead of final incubation at 70° C. for 30min to 3 hr, the sample would be amplified in a thermocycler for 30-40cycles of 94° C./40 sec, 55° C./90 sec (see Sec. II.B.). As soon as thelast PCR step is completed, the sample is washed 3 times in the washbuffer (4×SSC; 0.05% Tween 20) heated to 70° C., for 2 min. The signalis then observed.

Another alternative is to amplify the cDNA generated from the initial RTreaction by using the GeneAmp in situ PCR system 1000 and GeneAmp insitu PCR core kit (Perkin Elmer).

One step in situ RT-PCR on a fixed and permeabilized sample can beperformed using GeneAmp EZ rTth RNA PCR protocol in combination withGeneAmp in situ PCR system 1000 (Perkin Elmer). This method consist ofplacing 40-50 μl of EZ RNA PCR buffer mix (50 mM Bicine; 115 mMpotassium acetate; 8% (w/v) glycerol, pH 8.2; 300 μM dA, dG, dCTP; 165μM dTTP; 135 μM labeled dUTP; 5-10 U of rTth DNA polymerase; 2.5 mMMn(OAc)₂; 0.45-1 μM of htRNA-specific primers e.g. R7 and U3b onto afixed and permeabilized sample on a microscopic slide, and sealing itwith the silicone gastket and clip following the manufacturer's protocol(GeneAmp in situ PCR system 1000, Perkin Elmer). The sample is thenplaced in GeneAmp in situ PCR machine and heated for 120 sec at 94° C.,and then amplified for 30-40 cycles of 94° C./45 sec, and 60° C./45 sec.After the amplification, the sample is washed and visualized asdiscussed previously.

To reduce the background signals that can arise from directincorporation of fluorescent tags during the PCR amplication, indirectdetection that consist of PCR amplification using non-tagged dNTPsfollowed by in situ hybridization utilizing a tagged hybridization probespecific for the amplified product can be used. In this method, thesignal is amplified by any of the RT-PCR method described above withoutlabeled dNTPs or primers, and the amplified product is detected by insitu hybridization.

D. Application of Product Extension Primer to in Situ PCR

The success of in situ PCR depends on the prevention of the amplifiedproducts inside the cellular matrix from leaking out of the cell.Therefore, it is generally true that the PCR product smaller than 500 bpis not desirable for in situ PCR. In order to prevent the leakage of thePCR products smaller than 500 bp from the cellular matrix, incorporationof “bulky” dNTPs (e.g., biotin, fluorescent tag, digoxigenin labeleddUTP) into the PCR product is commonly used. Another way to prevent theleakage of a small products in in situ PCR would be to incorporateproduct extension primer into the in situ PCR protocol.

The method consists of using a primer that contains 3-4 6 bp repetitivesequence (e.g. [5′-TTTCCC-3′]₃₋₄) (SEQ ID NO:38) at the 5′ end, followedby a sequence that is specific for the target (see FIG. 4, primer 1), incombination with appropriate return primer (primer 2), and a thirdprimer that consist solely of the repetitive sequence (primer 3, e.g.[5′-TTTCCC-3′]₄); (SEQ ID NO:37 and 38) to amplify the specific in insitu PCR. The presence of the primer 3 will elongate the PCR product dueto the staggered-binding of the primer 3 to 3′-end of the target PCRproduct. The elongation of the PCR products can be induced by decreasingthe anealing temperature of the initial PCR condition.

For example, if the annealing temperature of the target sequence in theprimer 1 is 60° C., the sample will be initially amplified for 15-20cycles of 94° C./45° C. and 60° C./45° C., then it will be amplified for15-20 cycles of 94° C./45° C. and 50° C./45° C. Lowered annealingtemperature in the second PCR step will favor the generation of theelongated PCR products by increasing the chance of stagger-binding ofprimer 3 to the repetative sequences. The resulting elongated PCRproducts will be less prone to leakage through the cellular matrix, thusresulting in a better signal retention in in situ PCR analysis.

The foregoing examples describe various aspects of the invention and howcertain nucleic acids of the invention were made. The examples are notintended to provide an exhaustive description of the many differentembodiments of the invention encompassed by the following claims.

42 560 base pairs nucleic acid single linear RNA (genomic) 1 GGGUUGCGGAGGGUGGGCCU GGGAGGGGUG GUGGCCAUUU UUUGUCUAAC CCUAACUGAG 60 AAGGGCGUAGGCGCCGUGCU UUUGCUCCCC GCGCGCUGUU UUUCUCGCUG ACUUUCAGCG 120 GGCGGAAAAGCCUCGGCCUG CCGCCUUCCA CCGUUCAUUC UAGAGCAAAC AAAAAAUGUC 180 AGCUGCUGGCCCGUUCGCCC CUCCCGGGGA CCUGCGGCGG GUCGCCUGCC CAGCCCCCGA 240 ACCCCGCCUGGAGGCCGCGG UCGGCCCGGG GCUUCUCCGG AGGCACCCAC UGCCACCGCG 300 AAGAGUUGGGCUCUGUCAGC CGCGGGUCUC UCGGGGGCGA GGGCGAGGUU CAGGCCUUUC 360 AGGCCGCAGGAAGAGGAACG GAGCGAGUCC CCGCGCGCGG CGCGAUUCCC UGAGCUGUGG 420 GACGUGCACCCAGGACUCGG CUCACACAUG CAGUUCGCUU UCCUGUUGGU GGGGGGAACG 480 CCGAUCGUGCGCAUCCGUCA CCCCUCGCCG GCAGUGGGGG CUUGUGAACC CCCAAACCUG 540 ACUGACUGGGCCAGUGUGCU 560 11 base pairs nucleic acid single linear RNA 2 CUAACCCUAAC 11 2426 base pairs nucleic acid single linear DNA (genomic) 3GATCAGTTAG AAAGTTACTA GTCCACATAT AAAGTGCCAA GTCTTGTACT CAAGATTATA 60AGCAATAGGA ATTTAAAAAA AGAAATTATG AAAACTGACA AGATTTAGTG CCTACTTAGA 120TATGAAGGGG AAAGAAGGGT TTGAGATAAT GTGGGATGCT AAGAGAATGG TGGTAGTGTT 180GACATATAAC TCAAAGCATT TAGCATCTAC TCTATGTAAG GTACTGTGCT AAGTGCAATA 240GTGCTAAAAA CAGGAGTCAG ATTCTGTCCG TAAAAAACTT TACAACCTGG CAGATGCTAT 300GAAAGAAAAA GGGGATGGGA GAGAGAGAAG GAGGGAGAGA GATGGAGAGG GAGATATTTT 360ACTTTTCTTT CAGATCGAGG ACCGACAGCG ACAACTCCAC GGAGTTTATC TAACTGAATA 420CGAGTAAAAC TTTTAAGATC ATCCTGTCAT TTATATGTAA AACTGCACTA TACTGGCCAT 480TATAAAAATT CGCGGCCGGG TGCGGTGGCT CATACCTGTA ATCCCAGCAC TTTGGGAGGC 540CGAAGCGGGT GGATCACTTG AGCCCTGGCG TTCGAGACCA GCCTGGGCAA CATGGTGAAA 600CCCCCGTCTC TACTAAAAAC ACAAAAACTA GCTGGGCGTG GTGGCAGGCG CCTGTAATCC 660CAGCTACTCA GGAGGCTGAG ACACGAGAAT CGCTTGAACC CGGGAGCAGA GGTTGCAGTG 720AGCCGAGATC ACGCCACTAG ACTCCATCCA GCCTGGGCGA AAGAGCAAGA CTCCGTCTCA 780AAAAAAAAAA TCGTTACAAT TTATGGTGGA TTACTCCCCT CTTTTTACCT CATCAAGACA 840CAGCACTACT TTAAAGCAAA GTCAATGATT GAAACGCCTT TCTTTCCTAA TAAAAGGGAG 900ATTCAGTCCT TAAGATTAAT AATGTAGTAG TTACACTTGA TTAAAGCCAT CCTCTGCTCA 960AGGAGAGGCT GGAGAAGGCA TTCTAAGGAG AAGGGGGCAG GGTAGGAACT CGGACGCATC 1020CCACTGAGCC GAGACAAGAT TCTGCTGTAG TCAGTGCTGC CTGGGAATCT ATTTTCACAA 1080AGTTCTCCAA AAAATGTGAT GATCAAAACT AGGAATTAGT GTTCTGTGTC TTAGGCCCTA 1140AAATCTTCCT GTGAATTCCA TTTTTAAGGT AGTCGAGGTG AACCGCGTCT GGTCTGCAGA 1200GGATAGAAAA AAGGCCCTCT GATACCTCAA GTTAGTTTCA CCTTTAAAGA AGGTCGGAAG 1260TAAAGACGCA AAGCCTTTCC CGGACGTGCG GAAGGGCAAC GTCCTTCCTC ATGGCCGGAA 1320ATGGAACTTT AATTTCCCGT TCCCCCCAAC CAGCCCGCCC GAGAGAGTGA CTCTCACGAG 1380AGCCGCGAGA GTCAGCTTGG CCAATCCGTG CGGTCGGCGG CCGCTCCCTT TATAAGCCGA 1440CTCGCCCGGC AGCGCACCGG GTTGCGGAGG GTGGGCCTGG GAGGGGTGGT GGCCATTTTT 1500TGTCTAACCC TAACTGAGAA GGGCGTAGGC GCCGTGCTTT TGCTCCCCGC GCGCTGTTTT 1560TCTCGCTGAC TTTCAGCGGG CGGAAAAGCC TCGGCCTGCC GCCTTCCACC GTTCATTCTA 1620GAGCAAACAA AAAATGTCAG CTGCTGGCCC GTTCGCCCCT CCCGGGGACC TGCGGCGGGT 1680CGCCTGCCCA GCCCCCGAAC CCCGCCTGGA GGCCGCGGTC GGCCCGGGGC TTCTCCGGAG 1740GCACCCACTG CCACCGCGAA GAGTTGGGCT CTGTCAGCCG CGGGTCTCTC GGGGGCGAGG 1800GCGAGGTTCA GGCCTTTCAG GCCGCAGGAA GAGGAACGGA GCGAGTCCCC GCGCGCGGCG 1860CGATTCCCTG AGCTGTGGGA CGTGCACCCA GGACTCGGCT CACACATGCA GTTCGCTTTC 1920CTGTTGGTGG GGGGAACGCC GATCGTGCGC ATCCGTCACC CCTCGCCGGC AGTGGGGGCT 1980TGTGAACCCC CAAACCTGAC TGACTGGGCC AGTGTGCTGC AAATTGGCAG GAGACGTGAA 2040GGCACCTCCA AAGTCGGCCA AAATGAATGG GCAGTGAGCC GGGGTTGCCT GGAGCCGTTC 2100CTGCGTGGGT TCTCCCGTCT TCCGCTTTTT GTTGCCTTTT ATGGTTGTAT TACAACTTAG 2160TTCCTGCTCT GCAGATTTTG TTGAGGTTTT TGCTTCTCCC AAGGTAGATC TCGACCAGTC 2220CCTCAACGGG GTGTGGGGAG AACAGTCATT TTTTTTTGAG AGATCATTTA ACATTTAATG 2280AATATTTAAT TAGAAGATCT AAATGAACAT TGGAAATTGT GTTCCTTTAA TGGTCATCGG 2340TTTATGCCAG AGGTTAGAAG TTTCTTTTTT GAAAAATTAG ACCTTGGCGA TGACCTTGAG 2400CAGTAGGATA TAACCCCCAC AAGCTT 2426 20 base pairs nucleic acid singlelinear RNA modified_base /mod_base= cm modified_base /mod_base= ummodified_base /mod_base= cm modified_base /mod_base= OTHER /note= “N =2′-O-methyl-riboadenine” modified_base /mod_base= gm modified_base/mod_base= um modified_base /mod_base= um modified_base /mod_base= OTHER/note= “N = 2′-O-methyl-riboadenine” modified_base /mod_base= gmmodified_base 10 /mod_base= gm modified_base 11 /mod_base= gmmodified_base 12 /mod_base= um modified_base 13 /mod_base= ummodified_base 14 /mod_base= OTHER /note= “N = 2′-O-methyl-riboadenine”modified_base 15 /mod_base= gm modified_base 16 /mod_base= OTHER /note=“N = 2′-O-methyl-riboadenine” modified_base 17 /mod_base= cmmodified_base 18 /mod_base= OTHER /note= “N = 2′-O-methyl-riboadenine”modified_base 19 /mod_base= OTHER /note= “N = 2′-O-methyl-riboadenine”modified_base 20 /mod_base= OTHER /note= “N = 2′-O-methyl-riboadenine” 4NNNNNNNNNN NNNNNNNNNN 20 22 base pairs nucleic acid single linear RNAmodified_base /mod_base= cm modified_base /mod_base= gm modified_base/mod_base= cm modified_base /mod_base= cm modified_base /mod_base= cmmodified_base /mod_base= um modified_base /mod_base= um modified_base/mod_base= cm modified_base /mod_base= um modified_base 10 /mod_base= cmmodified_base 11 /mod_base= OTHER /note= “N = 2′-O-methyl-riboadenine”modified_base 12 /mod_base= gm modified_base 13 /mod_base= ummodified_base 14 /mod_base= um modified_base 15 /mod_base= OTHER /note=“N = 2′-O-methyl-riboadenine” modified_base 16 /mod_base= gmmodified_base 17 /mod_base= gm modified_base 18 /mod_base= gmmodified_base 19 /mod_base= um modified_base 20 /mod_base= ummodified_base 21 /mod_base= OTHER /note= “N = 2′-O-methyl-riboadenine”modified_base 22 /mod_base= gm 5 NNNNNNNNNN NNNNNNNNNN NN 22 22 basepairs nucleic acid single linear RNA modified_base /mod_base= gmmodified_base /mod_base= gm modified_base /mod_base= cm modified_base/mod_base= gm modified_base /mod_base= cm modified_base /mod_base= cmmodified_base /mod_base= um modified_base /mod_base= OTHER /note= “N =2′-O-methyl-riboadenine” modified_base /mod_base= cm modified_base 10/mod_base= gm modified_base 11 /mod_base= cm modified_base 12 /mod_base=cm modified_base 13 /mod_base= cm modified_base 14 /mod_base= ummodified_base 15 /mod_base= um modified_base 16 /mod_base= cmmodified_base 17 /mod_base= um modified_base 18 /mod_base= cmmodified_base 19 /mod_base= OTHER /note= “N = 2′-O-methyl-riboadenine”modified_base 20 /mod_base= gm modified_base 21 /mod_base= ummodified_base 22 /mod_base= um 6 NNNNNNNNNN NNNNNNNNNN NN 22 20 basepairs nucleic acid single linear DNA 7 CAGGCCCACC CTCCGCAACC 20 9 basepairs nucleic acid single linear DNA 8 CTAACCCTA 9 9 base pairs nucleicacid single linear DNA 9 CAAACCCAA 9 10 base pairs nucleic acid singlelinear DNA 10 CCAACCCCAA 10 10 base pairs nucleic acid single linear DNA11 CTCACCCTCA 10 39 base pairs nucleic acid single linear RNA 12UAGGGUUACU GAUGAGUCCG UGAGGACGAA ACAAAAAAU 39 37 base pairs nucleic acidsingle linear RNA 13 UUAGGGUCUG AUGAGUCCGU GAGGACGAAA GACAAAA 37 36 basepairs nucleic acid single linear RNA 14 UCUCAGUCUG AUGAGUCCGU GAGGACGAAAGGGUUA 36 36 base pairs nucleic acid single linear RNA 15 CCCGAGACUGAUGAGUCCGU GAGGACGAAA CCCGCG 36 20 base pairs nucleic acid single linearDNA modified_base /mod_base= OTHER /note= “N = 5′-phosphorylatedadenine” modified_base 20 /mod_base= OTHER /note= “N = adenine with3′-amino blocking group” 16 NTAGCGGCCG CAAGAATTCN 20 20 base pairsnucleic acid single linear DNA 17 TGAATTCTTG CGGCCGCTAT 20 12 base pairsnucleic acid single linear RNA modified_base /mod_base= um modified_base/mod_base= um modified_base /mod_base= OTHER /note= “N =2′-O-methyl-riboadenine” modified_base /mod_base= gm modified_base/mod_base= gm modified_base /mod_base= gm modified_base /mod_base= ummodified_base /mod_base= um modified_base /mod_base= OTHER /note= “N =2′-O-methyl-riboadenine” modified_base 10 /mod_base= gm modified_base 11/mod_base= i modified_base 12 /mod_base= OTHER /note= “N = biotinylatedinosine” 18 NNNNNNNNNN NN 12 12 base pairs nucleic acid single linearRNA modified_base /mod_base= OTHER /note= “N = 2′-O-methyl-riboadenine”modified_base /mod_base= um modified_base /mod_base= um modified_base/mod_base= gm modified_base /mod_base= gm modified_base /mod_base= gmmodified_base /mod_base= um modified_base /mod_base= um modified_base/mod_base= OTHER /note= “N = 2′-O-methyl-riboadenine” modified_base 10/mod_base= um modified_base 11 /mod_base= i modified_base 12 /mod_base=OTHER /note= “N = biotinylated inosine” 19 NNNNNNNNNN NN 12 17 basepairs nucleic acid single linear DNA 20 GGAAGGTCTG AGACTAG 17 17 basepairs nucleic acid single linear DNA 21 ATCTCCTGCC CAGTCTG 17 30 basepairs nucleic acid single linear DNA 22 GAGAAAAACA GCGCGCGGGG AGCAAAAGCA30 26 base pairs nucleic acid single linear DNA 23 GTTTGCTCTA GAATGAACGGTGGAAG 26 20 base pairs nucleic acid single linear DNA 24 CACCGGGTTGCGGAGGGAGG 20 21 base pairs nucleic acid single linear DNA 25 GGAGGGGCGAACGGGCCAGC A 21 18 base pairs nucleic acid single linear DNA 26TTAGGGTTAG GGTTAGGG 18 25 base pairs nucleic acid single linear DNA 27CAACGAACGG CCAGCAGCTG ACATT 25 20 base pairs nucleic acid single linearDNA modified_base /mod_base= OTHER /note= “N = 5′ phosphorylatedadenosine” 28 NTAGCGGCCG CTTGCCTTCA 20 28 base pairs nucleic acid singlelinear DNA 29 AGAAAAACAG CGCGCGGGGA GCAAAGCA 28 26 base pairs nucleicacid single linear DNA 30 TCTAACCCTA ACTGAGAAGG GCGTAG 26 9 base pairsnucleic acid single linear RNA 31 CUAACCCUA 9 9 base pairs nucleic acidsingle linear RNA 32 CCAACCCCA 9 17 base pairs nucleic acid singlelinear DNA 33 CCCAAACCCA AACCCAA 17 24 base pairs nucleic acid singlelinear DNA 34 ACCCAAACCC AAACCCAAAC CCAA 24 18 base pairs nucleic acidsingle linear DNA 35 CCCCAACCCC AACCCCAA 18 18 base pairs nucleic acidsingle linear DNA 36 CCAACCCCAA CCCCAACC 18 18 base pairs nucleic acidsingle linear DNA 37 TTTCCCTTTC CCTTTCCC 18 24 base pairs nucleic acidsingle linear DNA 38 CCAACCCCAA CCCCAACCTT TCCC 24 12 base pairs nucleicacid single linear DNA 39 TTAGGGTTAG GG 12 20 base pairs nucleic acidsingle linear RNA 40 CUCAGUUAGG GUUAGACAAA 20 22 base pairs nucleic acidsingle linear RNA 41 CGCCCUUCUC AGUUAGGGUU AG 22 22 base pairs nucleicacid single linear RNA 42 GGCGCCUACG CCCUUCUCAG UU 22

What is claimed is:
 1. A method for inhibiting telomerase activity inhuman cells, comprising contacting the human cells in vivo with anexogenous oligonucleotide in an amount sufficient to inhibit telomeraseactivity, the oligonucleotide comprising at least 10 consecutivenucleotides exactly complementary to a human telomerase RNA componentsequence, wherein the human telomerase RNA component comprises asequence located in an ˜2.5 kb HindIII-SacI insert of plasmid pGRN33(ATCC 75926), wherein the oligonucleotide does not consist of aplurality of 5′-TTAGGG-3′ telomeric repeat units, and wherein theoligonucleotide inhibits telomerase activity.
 2. The method of claim 1,wherein the cells are neoplastic cells.
 3. The method of claim 1 whereinthe oligonucleotide is a ribonucleic acid or a deoxyribonucleic acid. 4.The method of claim 1 wherein the oligonucleotide is a syntheticpolynucleotide.
 5. The method of claim 1 wherein the oligonucleotide isbetween 10 and 50 nucleotides long.
 6. The method of claim 5 wherein theoligonucleotide is between 25 and 50 nucleotides long.
 7. The method ofclaim 1 wherein the oligonucleotide comprises one or more non-naturallyoccurring nucleotides or nucleotide linkages.
 8. The method of claim 7wherein the oligonucleotide comprises a moiety selected from the groupconsisting of a methylphosphonate moiety, a C-5 propynyl moiety, and a2′-fluororibose sugar moiety or is selected from the group consisting ofa phosphorothioate polynucleotide, a 2′- O-methyl polynucleotide, apolyamide polynucleotide, and a polyamide nucleic acid polynucleotide.9. The method of claim 1, wherein the exogenous oligonucleotide ispresent in a pharmaceutical formulation.
 10. A method for inhibitingtelomerase activity in human cells, comprising contacting the humancells in vivo with an exogenous oligonucleotide in an amount sufficientto inhibit telomerase activity, the oligonucleotide comprising more than12 consecutive nucleotides exactly complementary to a human telomeraseRNA component sequence, wherein the human telomerase RNA componentcomprises a sequence located in an ˜2.5 kb HindIII-SacI insert ofplasmid pGRNT33 (ATCC 75926), and wherein the oligonucleotide inhibitstelomerase activity.
 11. A method for inhibiting telomerase activity inhuman cells, comprising contacting the human cells in vivo with anexogenous oligonucleotide in an amount sufficient to inhibit telomeraseactivity, the oligonucleotide comprising at least 10 consecutivenucleotides exactly complementary to SEQ ID NO:1, wherein theoligonucleotide does not consist of a plurality cif 5′-TTAGGG-3′telomeric repeat units, and wherein the oligonucleotide inhibitstelomerase activity.
 12. The method of claim 11, wherein the cells areneoplastic cells.
 13. The method of claim 11 wherein the oligonucleotideis a ribonucleic acid or a deoxyribonucleic acid.
 14. The method ofclaim 11 wherein the oligonucleotide is a synthetic polynucleotide. 15.The method of claim 11 wherein the oligonucleotide is between 10 and 50nucleotides long.
 16. The method of claim 11 wherein the oligonucleotidecomprises one or more non-naturally occurrng nucleotides or nucleotidelinkages.
 17. The method of claim 11, wherein the exogenousoligonucleotide is present in a pharmaceutical formulation.
 18. A methodfor inhibiting telomerase activity in human cells, comprising contactingthe human cells in vivo with an exogenous oligonucleotide in an amountsufficient to inhibit telomerase activity, the oligonucleotidecomprising more than 12 consecutive nucleotides exactly complementary toSEQ ID NO:1, wherein the oligonucleotide inhibits telomerase activity.